Bacterial ribonucleic acid cell wall compositions and methods of making and using them

ABSTRACT

The present invention relates to novel bacterial and mycobacterial compositions containing RNA and cell walls, and methods for making and using these compositions. These compositions have immune stimulating and anti-cancer activity.

PRIOR RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 13/701,954 filed Dec. 4, 2012, which is a nationalphase of PCT/IB2011/054539 filed Oct. 13, 2011, which claims the benefitof priority to U.S. provisional patent application 61/392,498 filed Oct.13, 2010, and U.S. provisional patent application 61/393,589 filed Oct.15, 2010, each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to novel compositions comprising bacterialRNA and bacterial cell walls and methods for making and using thesecompositions. These compositions have immune stimulating and anti-canceractivity.

BACKGROUND OF THE INVENTION

The treatment of cancer continues to be a problem for clinical andveterinary medicine. The treatment regimens available today includesurgery, radiation, chemotherapy, immunotherapy (including autologousand heterologous cell therapy) or combinations thereof

Surgery often fails due to tumor tissue being unrecognized and notremoved. Radiation and chemotherapy also frequently fail, and the sideeffects of the treatments often decrease the quality of life forpatients. Surgery and chemotherapy are associated with significant andoften non-specific suppression of the immune system (Hammer et al., Eur.Surg. Res., 1992 24:133-137; Joos and Tam, Proc. Am. Thorac. Soc., 20052:445-448). This immune suppression is often associated with theoccurrence of opportunistic infections, as exemplified by the known highrate of infectious complications in individuals undergoing high-dosewhole-body irradiation (Gil et al., Infection, 2007 35:421-427).Radiation, surgery and chemotherapy are additionally associated withmultilineage hematopoietic and myeloid suppression (myelosuppression)such as, but not limited, to leucopenia, neutropenia, thrombocytopeniaand/or anemia (Montoya. J. Infus. Nurs. 2007 30:168-172). Theseconditions may be life threatening for patients. Chemotherapy is oftencompromised by the presence of or subsequent development of resistance,which can often span different classes of drugs (multidrug resistance).

Immunotherapy for cancer has been employed for many years. One of thefirst immune treatments was a mixed bacterial vaccine (Coley's vaccine),the active ingredient of which is bacterial lipopolysaccharide. Itshould be noted that regulatory authorities strive to limit or eliminatethe presence of lipopolysaccharide from pharmaceutical agents due tounwanted and often toxic effects. More recently, mixtures of irradiatedmalignant melanoma cells have been used to induce immune responses inpatients with malignant melanoma, which increased survival in severalpatients (Morton, et al. Ann. Surg. 1992, 216:463-482). One majorbenefit offered by immune therapy (immunotherapy) is that it is notgenerally associated with the side effects of surgery, radiation orchemotherapy. In three studies using dendritic cell immunotherapy inpatients with cancer, minimal to no side effects were reported (Hsu, etal. Nature Medicine, 1996 2:52-58; Murphy, et al. The Prostate, 199629:371-380; Nestle, et al. Nature Medicine, 1998, 4(3):328-332).

Mycobacterial cell walls are known to stimulate host immune defensemechanisms (both innate through the interaction with pathogen associatedmolecular pattern receptors—PAMPs, and acquired through the presence ofimmunogenic molecular species). Immunotherapy using whole, viablemycobacteria is used clinically in the treatment of bladder cancer. Themycobacterium bacillus Calmette-Guérin (BCG), an attenuated strain ofMycobacterium bovis, is repeatedly instilled into the bladder ofindividuals with bladder cancer, where possible and preferably in anadjuvant setting following tumor removal by surgery (as described in forexample the European Association of Urology Guidelines 2007 edition,pages 8-9). Its use however is associated with a range of adverse sideeffects related to its viable nature (Koya, et al. J. Urology 2006,175:2004-2010) as well as an often low clinical efficacy and duration ofresponse rate, especially in patients who experience treatment relapse(Witjes and Hendricksen. Eur. Urol. 2008, 53:24-26). Its use for thetreatment of other cancers is contra-indicated because it contains livemycobacteria, and can give rise to fatal systemic infections (Orifice,et al. Tumori 1978, 64:437-443). Immunotherapy of cancer using intactbut inactivated mycobacteria has been attempted using the mycobacteriumMycobacterium vaccae, but no definitive long-term survival following itsuse has so far been identified in clinical studies (see Stanford et al.,Eur. J. Cancer 2008, 44:224-227). It is clear that intact mycobacteria,whether viable or inactivated, do not represent the most effective formof immunotherapy for the treatment of cancer.

Immunotherapy utilizing bacterial cell walls and bacterial extracts hasbeen extensively evaluated in animal tumor models, in patients sufferingfrom cancer (U.S. Pat. Nos. 4,503,048, 5,759,554 and 6,326,357), and astreatments for infectious diseases, such as bacterial and viralinfections (U.S. Pat. Nos. 3,172,815, and 4,744,984).

Mycobacterial cell wall compositions with immune stimulant andanticancer activity (for example as described in U.S. Pat. Nos.4,503,048, 5,759,554 and 6,329,347 or in Ribi et al., J. Bacteriol.1965, 91:975-983) suffer from the disadvantage that biological reagentsand materials, chemical reagents, solvents or diluents and enzymatictreatments are required for their preparation, with the potential fornoxious chemical and foreign protein contamination. Moreover, it hasbeen reported that in order to obtain optimal anticancer activity withhighly purified mycobacterial cell walls (essentially consisting of thecell wall skeleton following extensive chemical and enzymatictreatments) formulation as oil emulsions is required (Yarkoni and Rapp,Cancer Res., 1979 39:535-7). Oil emulsions containing mycobacterial cellwalls are often physically unstable and are difficult to preparereproducibly, and can be toxic to the recipient because of thewell-known potential to induce hypersensitivity reactions. Mycobacterialcell walls containing biologically active complexed DNA that possessboth immunotherapeutic and anticancer activity and that do not depend onthe presence of oil have also been described (U.S. Pat. No. 6,326,357),but these again suffer from the disadvantage that chemical and enzymatictreatments are required for their preparation, with the potential fornoxious chemical and foreign protein contamination. In addition, usingsuch compositions it has not proven possible to preferentially optimizeeither the immunotherapeutic activity or the anticancer activity.

It is recognized by those of ordinary skill in the art that disruptionof microorganisms can be achieved using small sample volumes andrefrigerated pressure cells (such as the Sorvall pressure cell) at highpressures of between 40,000-45,000 pounds per square inch (PSI,equivalent to 276-317 mPa) (see Ribi et al., J. Bacterial., 1966,91:975-983). Such processes are time consuming, inefficient, and of lowvolume, and are additionally hampered by the current unavailability ofthis type of equipment. More efficient processes that use high pressurehomogenization have been described for the isolation of proteins (asinclusion bodies) from genetically engineered microorganisms (seePeternel and Komel: Isolation of biologically active nanomaterial[inclusion bodies] from bacterial cells. Microbial Cell Factories 20109:66). It is however recognized by those of skill in the art thatGram-positive organisms are resistant to such processes by virtue oftheir peptidoglycan content and structure (see Diels and Michaels: Highpressure homogenization as a non-thermal technique for the inactivationof microorganisms. Crit. Rev. Microbiol., 2006; 32:201-216). The use oftechniques to minimize the number of homogenization cycles is alsotaught by those skilled in the art (see Bailey et al., Improvedhomogenization of recombinant Escherichia coli following pretreatmentwith guanidinium chloride: Biotech. Prog., 1995; 11:533-539). The use ofsuch procedures is in fact clearly designed to remove cell wallfragments, not preserve them. The presence of nucleic acids such as DNAusing high pressure disruption techniques is additionally taught as acontaminating material to be removed, not preserved (see Rathore et al.,Analysis for residual host cell proteins and DNA in process streams of arecombinant protein product expressed in Escherichia coli cells: J.Pharm. Biomed. Anal., 2003; 32:1199-1211). What is needed are newprocedures for the preparation of new bacterial nucleic acidcompositions that use working volumes that are scalable, range fromseveral mL to multi-liter volumes, and result in the efficientproduction of new bacterial nucleic acid and cell wall compositions.

It is known that mycobacterial cell walls and components thereof canstimulate and activate macrophages, monocytes and dendritic cells toproduce bioactive molecules that can initiate, accelerate, amplify andstimulate responsive cells of the immune system such that an immunestimulatory effect is achieved. These bioactive molecules include, butare not limited to, hematopoietic and myeloid growth factors, cytokinesand chemokines.

Growth factors are proteins that bind to receptors on a cell surface,with the primary result of activating cellular proliferation and/ordifferentiation. Cytokines are a unique family of regulatory proteins.Secreted primarily from cells of the immune system such as but notlimited to leukocytes and acting as intercellular mediators, cytokinesstimulate the humoral and cellular immune response, as well as theactivation of phagocytic cells. Cytokines that are secreted fromlymphocytes are termed lymphokines, whereas those secreted by monocytesor macrophages are termed monokines Many of the lymphokines are alsoknown as interleukins (IL), since they are not only secreted byleukocytes but also able to affect the cellular responses of leukocytes.Chemokines are a class of cytokines that have the ability to attract andactivate leukocytes, especially in response to infections (a processtermed chemotaxis). They can be divided into at least three structuralbranches: c (chemokines, c), cc (chemokines, cc), and cxc (chemokines,cxc), according to variations in a shared cysteine motif (Johrer et al.Exp. Opin. Biol. Ther. 2008 8:269-290.). The harmful effects ofchemotherapeutic agents or radiation therapy on the production of thecells of the immune system that are responsible for producing thesehematopoietic and myeloid growth factors, cytokines and chemokinesresults in increased susceptibility to opportunistic infections.

Cancer (of which there are over 100 diseases) is an aberrant netaccumulation of atypical cells, which results from uncontrolled celldivision, an insufficiency of or defective apoptosis, or a combinationof the two. Mutations in apoptosis-related genes such as, but notlimited to, Fas, TNFR1 and p53/p21 have each been implicated in thepathogenesis of cancers (Levine, A. Cell 88:323-331, 1997; Fisher, D.Cell 78:529-542, 1994). Aberrant apoptosis is important not only to thepathogenesis of cancers, but also to a cancer's likelihood of resistanceto many anti-cancer therapies.

Resistance to apoptosis induction has emerged as an important categoryof multiple drug resistance (MDR), one that likely explains asignificant proportion of treatment failures. MDR, the simultaneousresistance to structurally and functionally unrelated classes ofchemotherapeutic agents, can be both inherent and acquired. That is,some cancers never respond to therapy, whereas other cancers, initiallysensitive to therapy, subsequently develop drug resistance through theselection of resistant clones. As chemotherapeutic agents rely primarilyon an induction of apoptosis in cancer cells for their therapeuticeffect, drug resistance, which diminishes the effectiveness ofchemotherapeutic agents, leads directly or indirectly to reducedapoptosis and is generally associated with poor clinical prognosis in avariety of cancers.

Prior art anti-cancer agents have proven to be less than adequate forclinical applications. Many of these agents are inefficient (Bischoff etal. Science 274:373-376, 1996) or toxic, have significant side effects(Lamm et al. Journal of Urology 153:1444-1450, 1995), result in thedevelopment of drug resistance or immune sensitization andhypersensitivity reactions, and are debilitating for the recipient.

Therefore, there is a need for novel therapeutic compositions thatstimulate responsive cells of the immune system to produce cytokines,chemokines and hematopoietic and myeloid growth factors, inhibitproliferation of cancer cells and induce apoptosis in cancer cells.These therapeutic compositions should be useful as an anti-cancer agentin their own right as well as having an adjuvant activity with respectto other anti-cancer agents. This therapeutic composition should beuseful in preventing or treating myelosuppression associated withcancer, surgery, radiation or chemotherapy. Moreover, such a therapeuticcomposition should be simple and relatively inexpensive to prepare, itsactivity should be reproducible among preparations, its activity shouldremain stable over time, and its effects on cancer cells should beachievable with dose regimens that are associated with minimal adversereactivity and toxicity. In addition, there is a need for methods ofmanufacturing novel therapeutic compositions that are efficient and thatdo not result in the presence of enzymes or chemicals used in theirpreparation.

There is also a need for a novel therapeutic composition that treats,prevents, abates or ameliorates autoimmune disorders, inflammatory orinfectious disease, myelosuppression or hematopoietic and myeloidabnormalities. The therapeutic composition should be useful as anadjuvant with other therapeutic agents. Moreover, such a therapeuticcomposition should be simple and relatively inexpensive to prepare, itsactivity should be reproducible among preparations, its activity shouldremain stable over time, and its effects should be achievable with doseregimens that are associated with minimal toxicity.

SUMMARY OF THE INVENTION

The present invention satisfies the above needs by providing novelcompositions comprising ribonucleic acid (RNA) and cell walls (definedas RNC herein) which are derived from bacteria or mycobacteria. Thepresent invention satisfies the above needs by providing novelcompositions comprising bacterial ribonucleic acid (RNA) and bacterialcell walls (defined as BRNC) and methods of making and using thesecompositions. The present invention also satisfies the above needs byproviding novel compositions comprising mycobacterial RNA andmycobacterial cell walls (defined as MRNC) and methods of making andusing these compositions. These novel compositions comprise bacterial ormycobacterial RNA in the form of oligoribonucleotides andpolyribonucleotides wherein the RNA can exist as single strands (ss),double strands (ds), or single-double stranded hybrid strands (hs),isolated from bacteria or mycobacteria and bacterial or mycobacterialcell walls, wherein the RNA is formulated in a pharmaceuticallyacceptable vehicle or complexed (where the term complex is used todescribe the chemical association of two or more molecular species) to apharmaceutically acceptable carrier system. Although pharmaceuticalcarrier systems known to those of skill in the art can be used, in oneembodiment the carrier system is based on bacterial cell walls ormycobacterial cell walls. The compositions of the present invention mayalso contain intact bacterial or mycobacterial cells, the content ofwhich is selected for the desired therapeutic intent.

The compositions of the present invention possess anti-cancer activity.The compositions of the present invention also contain immune systemstimulating activity. The compositions of the present invention also actas adjuvants to other therapeutic modalities.

Three different names are used in the present application to describethese novel compositions. First, the term bacterial RNA composition(BRNC) is used generally to describe the compositions made fromdifferent species of bacteria. BRNC may be prepared from differentGram-negative or Gram-positive bacteria with the methods disclosedherein. Second, the term mycobacterial RNA composition (MRNC) is used todescribe the compositions made from different mycobacteria. In differentembodiments, MRNC may be prepared from any mycobacterium. Third, inother embodiments, specific mycobacterial species, mycobacterialcomplexes or mycobacterial strains are used to prepare MRNC. Examples ofbut not limited to these are Mycobacterium avium (including sub-speciesparatuberculosis, commonly termed MAP), Mycobacterium bovis BCG,Mycobacterium phlei, Mycobacterium smegmatis, or Mycobacterium vaccaewith the methods disclosed herein. It will be recognized by one of skillin the art that the methods of manufacturing the compositions describedherein are applicable to the manufacture of MRNC from mycobacteria otherthan those described in the detailed description and examples.

The term Mycobacterial RNA composition (MpRNC) is used to describe thecompositions comprising RNA and cell walls made from Mycobacteriumphlei. The terms Mycobacterium bovis strain BCG RNA composition (MbRNC),Mycobacterium smegmatis RNA composition (MsRNC), Mycobacterium aviumsubspecies paratuberculosis (MAP) RNA composition (MapRNC) andMycobacterium vaccae RNA composition (MvRNC) are used to describe thecompositions comprising RNA and cell walls made from Mycobacterium bovisBCG, Mycobacterium smegmatis, Mycobacterium avium subspeciesparatuberculosis and Mycobacterium vaccae, respectively. In eachcomposition, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC, RNA isisolated from mycobacteria using a procedure that results in anefficient production of oligoribonucleotides and polyribonucleotides.

In another embodiment, each MRNC, MpRNC, MbRNC, MsRNC, MapRNC or MvRNC,may also contain different amounts of intact mycobacterial cells. Intactcells may be present in MRNC, MpRNC, MbRNC, MsRNC, MapRNC or MvRNC inamounts of from approximately 0-99%, 0.05-95%, 0.07-50%, 0.1-20% or0.19-19% by weight, or in an amount within any of these ranges, or lessthan 0.75% or less than 0.2% by weight.

In another embodiment, these novel compositions possess an RNA content(which can range from approximately 30% to 100% of the total nucleicacid content) in the form of oligoribonucleotides andpolyribonucleotides that are 2 to 4000 bases in length. In anotherembodiment, these novel compositions possess an oligoribonucleotide andpolyribonucleotide content (approximately 30-100% of the total nucleicacid content) in the form of oligoribonucleotides andpolyribonucleotides that are 2 to 150 bases in length. In yet anotherembodiment, these novel compositions possess an oligoribonucleotide andpolyribonucleotide content (approximately 30-100% of the total nucleicacid content) in the form of oligoribonucleotides andpolyribonucleotides greater than 150, or 151 to 4000 bases in length. Inanother embodiment, these novel compositions possess anoligoribonucleotide and polyribonucleotide content (approximately30-100% of the total nucleic acid content) mostly in the form ofoligoribonucleotides and polyribonucleotides that are 20 to 40 bases inlength.

Anti-cancer activity of the present compositions is maximized when thereis minimal content of intact mycobacterial cells. Immune stimulatoryactivity is maximized when there is a content of intact mycobacterialcells in the range of 5-50% w/w. The desired content of intactmycobacterial, Mycobacterium phlei, Mycobacterium bovis BCG,Mycobacterium smegmatis, Mycobacterium avium subspecies paratuberculosisor Mycobacterium vaccae cells can be achieved by controlling themanufacturing process as detailed in the examples, or by adding intactmycobacterial, Mycobacterium phlei, Mycobacterium bovis BCG,Mycobacterium smegmatis, Mycobacterium avium subspecies paratuberculosisor Mycobacterium vaccae cells to MRNC, MpRNC, MbRNC, MsRNC, MapRNC orMvRNC. MRNC may be used alone as a composition prepared from onemycobacterial species, strain, sub-strain or complex, or in combinationwith MRNC from other mycobacterial species, strain, sub-strain orcomplex. For example, a combination of MpRNC and MvRNC will give optimalNOD2 and TLR2 activation for immune stimulation and cytokine induction.Similarly, a combination of MpRNC and MvRNC will give optimal anticanceractivity and immune stimulation.

These new compositions possess immune stimulatory, anti-cancer andhematopoietic and myeloid stem cell growth factor stimulating activity.As opposed to prior art preparations, these new compositions containoligoribonucleotides and polyribonucleotides (such as but not limited tosingle strands (ss), double strands (ds) or mixtures thereof), andcontain levels of intact mycobacteria appropriate for the intendedapplication. Also, as opposed to prior art, where combinations ofsynthetic agents are required (see Uehara et al., Muramyldipeptide anddiaminopimelic acid-containing desmuramylpeptides in combination withchemically synthesized Toll-like receptor agonists synergisticallyinduced production of interleukin-8 in a NOD2- and NOD-dependent manner,respectively, in human monocytic cells in culture: Cell. Microbiol.,2005; 7:53-61), these new compositions have the ability to activate boththe nucleotide-binding oligomerization domain 2 (NOD2) and Toll-likereceptor 2 (TLR2) receptors, thus demonstrating an unexpectedbifunctional agonist activity for immune system receptors that is usefulfor stimulation of the immune system, stimulating hematopoietic andmyeloid stem cell proliferation and in treating cancer in bothprophylactic or therapeutic treatment regimens. In differentembodiments, these compositions are effective in inducing a response inresponsive cells of an animal's immune system, in inducing cell cyclearrest or apoptosis and inhibiting cellular proliferation. Thesecompositions induce responsive cells of the immune system to producecytokines, chemokines and hematopoietic and myeloid stem cell growthfactors.

MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are relatively inexpensiveto prepare and their activity is reproducible among preparations. MRNC,MpRNC, MbRNC, MsRNC, MapRNC and MvRNC remain stable and effective overtime and at dose regimens that are associated with minimal toxicity.

In one embodiment of the invention, MRNC, MpRNC, MbRNC, MsRNC, MapRNCand MvRNC, are prepared from mycobacteria or Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis or Mycobacterium vaccae respectively, asfollows: the mycobacteria are grown and harvested. The mycobacteria aredisrupted to remove as required intact mycobacteria via the utilizationof high-pressure homogenization followed by a centrifugation process toeliminate as required any residual intact mycobacterial cells.Importantly, use is made of RNase-free and or non-specific nuclease-freereagents to optimize recovery of the RNC.

Low relative centrifugal force is used to remove intact mycobacteria asrequired. Oligoribonucleotides and polyribonucleotides can then beisolated using conventional extraction techniques, for exampleguanidinium thiocyanate-phenol-chloroform extraction, or a high relativecentrifugal force is applied to the supernatant after removal of intactmycobacteria to isolate oligoribonucleotides and polyribonucleotidesthat remain formulated with mycobacterial cell walls after cellulardisruption. Importantly, reagents that do not contain nucleasecontamination (non-specific endo- and exo-nucleases or ribonucleases)are used to eliminate or minimize RNA degradation and thereby optimizeyield during the preparation steps.

MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are effective therapeuticagents in preventing, treating, lessening the impact of and eliminatinga variety of diseases including, but not limited to, malignant,autoimmune and immunodeficiency diseases, myelosuppression andhematopoietic and myeloid abnormalities. MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC are particularly useful for treating diseases andprocesses mediated by undesired and uncontrolled cell proliferation,such as cancer. These compositions are also effective as adjuvants toenhance the effectiveness of other anti-cancer agents. Such anti-canceragents include, but are not limited to, drugs, immune stimulants,antigens, antibodies, vaccines, radiation, chemotherapeutic agents,genetic, biologically engineered and chemically synthesized agents, andagents that target cell death molecules for activation or inactivation,agents that inhibit proliferation of cancer cells, and that induceapoptosis in cancer cells. MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCare also effective for the prevention or treatment of myelosuppression(monocytopenia and or neutropenia) associated with the treatment ofcancer or cancer by itself, and for the prevention or treatment ofdiverse hematopoietic and myeloid abnormalities associated withmedications or diseases, such as, but not limited to the acquiredimmunodeficiency syndrome (AIDS), myelodysplastic syndromes, autoimmunediseases, end-stage renal diseases or viral infections.

MRNC, MpRNC, MbRNC, MsRNC, MapRNC or MvRNC, in a pharmaceuticallyacceptable carrier, may be administered to an animal or human in adosage effective to stimulate a response in responsive cells of theimmune system, and to inhibit proliferation of and induce apoptosis inresponsive cells. MRNC, MpRNC, MbRNC, MsRNC, MapRNC or MvRNC can beadministered by methods including, but not limited to, suspension inaqueous formulations, in creams and gels, by emulsification in oil orother hydrophobic liquid formulations, enclosure in liposomes, andcomplexation with natural or artificial carriers, with tissue- orcell-specific ligands or with tissue- or cell-specific antibodies.

In one embodiment, the present invention provides novel compositionscomprising, BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC or MvRNC, andmethods of making these compositions.

In another embodiment, the present invention provides novelmanufacturing procedures for the preparation of BRNC, MRNC, MpRNC,MbRNC, MsRNC, MapRNC or MvRNC compositions that optimize the content ofcarbon, nitrogen and iron sources in the cultivation media for themanufacture of mycobacterial cell mass.

In another embodiment, the present invention provides novel syntheticmedia useful for growing mycobacterial cell mass.

In yet another embodiment, the present invention provides novelmanufacturing procedures for the preparation of BRNC, MRNC, MpRNC,MbRNC, MsRNC, MapRNC or MvRNC compositions that optimize the content ofcarbon, nitrogen and iron in the cultivation media and that eliminatethe requirement for exogenous biological materials for the manufactureof mycobacterial cell mass, and that eliminate the use exogenousbiological or chemical agents during the down-stream manufacturingprocedure.

In still another embodiment, the present invention provides noveltherapeutic BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC or MvRNC, in apharmaceutically acceptable carrier.

In a further embodiment, the present invention provides therapeuticcompositions comprising BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC orMvRNC, and methods of using these therapeutic compositions to induce atherapeutic response in responsive cells of an animal or a human.

In another embodiment, the present invention provides a method tostimulate responsive cells of the immune system by administering thesetherapeutic compositions to an animal or a human.

In yet another embodiment, the present invention provides a method tostimulate responsive cells of the immune system to produce bioactivemolecules such as cytokines, chemokines, interleukins and/orhematopoietic and myeloid growth factors by administering thesetherapeutic compositions to an animal or a human.

In a further embodiment, the present invention provides a method that iseffective to treat a disease in an animal or human by administeringthese therapeutic compositions to an animal or a human.

In still another embodiment, the present invention provides a methodthat is effective to treat cancer in an animal or human by administeringthese therapeutic compositions to an animal or a human.

In another embodiment, the present invention provides a composition andmethod that inhibits proliferation of responsive cells of an animal.

In another embodiment, the present invention provides a composition andmethod that induces apoptosis in responsive cells of an animal.

In yet another embodiment, the present invention provides compositionand method that is effective as an adjuvant to other anti-cancertherapies.

In still another embodiment, the present invention provides acomposition and method that is effective as an adjuvant to other immunestimulatory therapies.

In a further embodiment, the present invention provides a compositionand method that is effective for the treatment of immunodeficiencydiseases.

In yet another embodiment, the present invention provides a compositionand method that is effective for the prevention or treatment ofmyelosuppression (leucopenia, neutropenia, thrombocytopenia or anemia)or hematopoietic and myeloid abnormalities.

In still another embodiment, the present invention provides acomposition and method that is effective as an adjuvant to othertherapies for the prevention or treatment of myelosuppression orhematopoietic and myeloid abnormalities.

In another embodiment, the present invention provides a method that iseffective to treat an autoimmune disease in an animal or human byadministering these therapeutic compositions to an animal or a human.

In yet another embodiment, the present invention provides a method ofmanufacturing that does not result in the presence of chemicals orenzymes in the composition.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiment and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-F. Transmission electron micrographs (TEM) of Mycobacteriumphlei (FIG. 1A), MCWE (FIG. 1B), MCC (FIG. 1C), MpRNC Low (FIG. 1D),MpRNC Intermediate (FIG. 1E) and MpRNC High (FIG. 1F). The bars are 1 μmfor FIG. 1A, FIG. 1B and FIG. 1C, and 2 μm for FIG. 1D, FIG. 1E and FIG.1F.

FIGS. 2A-B. Electrophoretic analysis of nucleic acid oligonucleotidelength. FIG. 2A. RNA ladder standard and MpRNC Intermediate prior toautoclaving using the RNA nano 6000 kit. FIG. 2B. MpRNC containingvarying proportions of autoclaved M. phlei cells (MpRNC High-, MpRNCLow- or and MpRNC Intermediate-) following autoclaving using the smallRNA kit (The peak at 4 nucleotides is the internal oligonucleotidestandard and is not associated with nucleic acids in the MpRNC.FU=fluorescence units, nt=nucleotide length.

FIGS. 3A-D. RNA and DNA content of MpRNC containing varying proportionsof intact autoclaved M. phlei cells (FIG. 3A High, FIG. 3B Intermediate,or FIG. 3C Low) and autoclaved Mycobacterium phlei (FIG. 3D) asdetermined before and after RNase-A treatment.

FIGS. 4A-B. RNase-susceptible nucleic acid (NA) in MpRNC, MbRNC, MsRNCand MvRNC. FIG. 4A: Urea-PAGE gel electrophoresis of extractedcontrol-treated and RNase-treated nucleic acids; FIG. 4B: The proportionof RNA in the extracted nucleic acids as determined by scanningdensitometry.

FIGS. 5A-B. Mycolic acid profile of MpRNC Intermediate. FIG. 5A: Mycolicacid profile using behenic acid standard for quantification ofextractable lipids; FIG. 5B: Mycolic acid profile using low and highmycolic acid carbon number standards (estimated to be ˜C40 and ˜C110)for quantification of saponifiable lipids.

FIG. 6. NOD2 activation by mycobacterial RNC. HEK293 cells expressingNOD2 were used to determine the NOD2 agonist activity of RNC preparedfrom 4 mycobacterial species—Mycobacterium phlei, Mycobacterium bovisstrain BCG, Mycobacterium smegmatis, and Mycobacterium vaccae. NOD2activation results in NF-κB-driven secretory embryonic alkalinephosphatase (SEAP) induction. Following hydrolysis of the substrate bySEAP in the cell culture supernatant, enzymatic activity is expressed asthe O.D. at 630 nm, and is proportional to NOD2 activation. Mean±oftriplicate determinations.

FIG. 7. TLR2 activation by mycobacterial RNC. HEK293 cells expressingthe TLR2 receptor were used to determine the TLR2 agonist activity ofMRNC prepared from 4 mycobacterial species—Mycobacterium phlei,Mycobacterium bovis strain BCG, Mycobacterium smegmatis, andMycobacterium vaccae. TLR2 activation results in NF-κB-driven SEAPinduction. Following hydrolysis of the substrate by SEAP in the cellculture supernatant, enzymatic activity is expressed as the O.D. at 630nm, and is proportional to NOD2 activation. Mean±of triplicatedeterminations.

FIGS. 8A-B. Stimulation of IL-10 and IL-12 production in humanperipheral blood mononuclear cells (PBMC) by MpRNC Intermediatecontaining varying proportions of intact autoclaved Mycobacterium.phlei. IL-10 (FIG. 8A) and IL-12p40 (FIG. 8B) subunit production byhuman PBMC following treatment with MpRNC and autoclaved Mycobacteriumphlei was determined by ELISA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compositions comprising ribonucleicacid (RNA) and cell walls (defined as RNC herein) which are derived frombacteria or mycobacteria. The present invention provides novelcompositions comprising bacterial RNA and bacterial cell walls (definedas BRNC) and methods of making and using these compositions. The presentinvention also provides novel compositions comprising mycobacterial RNAand mycobacterial cell walls (defined as MRNC) and methods of making andusing these compositions. These compositions can be formulated withcarriers, and/or cells. These novel compositions comprise cell wallsfrom bacteria or mycobacteria and RNA in the form ofoligoribonucleotides and polyribonucleotides from bacterial ormycobacterial RNA wherein the oligoribonucleotides andpolyribonucleotides can exist as single strands (ss), double strands(ds), or single-double stranded hybrid strands (hs). These compositionscan be formulated in a pharmaceutically acceptable vehicle or complexed(where the term complex is used to describe the chemical association of2 or more molecular species) to a pharmaceutically acceptable carriersystem. The compositions of the present invention may also containintact bacterial or mycobacterial cells, the content of which isoptimized for the desired therapeutic intent.

These new compositions possess immune stimulatory, anti-cancer andhematopoietic and myeloid stem cell growth factor stimulating activity.As opposed to prior art preparations, these new compositions containoligoribonucleotides and polyribonucleotides (such as but not limited tosingle strands (ss), double strands (ds) or mixtures thereof), andoptionally contain selected levels of intact mycobacteria appropriatefor the intended application. Also, as opposed to prior art, wherecombinations of synthetic agents are required (see Uehara et al.,Muramyldipeptide and diaminopimelic acid-containing desmuramylpeptidesin combination with chemically synthesized Toll-like receptor agonistssynergistically induced production of interleukin-8 in a NOD2- andNOD-dependent manner, respectively, in human monocytic cells in culture:Cell. Microbiol., 2005; 7:53-61), these new compositions have theability to activate both the nucleotide-binding oligomerization domain 2(NOD2) and Toll-like receptor 2 (TLR2) receptors, thus demonstrating anunexpected bifunctional agonist activity for immune system receptorsthat is useful for stimulation of the immune system, stimulatinghematopoietic and myeloid stem cell proliferation and in treating cancerin both prophylactic or therapeutic treatment regimens. In differentembodiments, these compositions are effective in inducing a response inresponsive cells of an animal's immune system, in inducing cell cyclearrest or apoptosis and inhibiting cellular proliferation. Thesecompositions induce responsive cells of the immune system to producecytokines, chemokines and hematopoietic and myeloid stem cell growthfactors.

MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are effective therapeuticagents in preventing, treating, lessening the impact of and eliminatinga variety of diseases including, but not limited to, malignant,autoimmune and immunodeficiency diseases, myelosuppression andhematopoietic and myeloid abnormalities. MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC are particularly useful for treating diseases andprocesses mediated by undesired and uncontrolled cell proliferation,such as cancer. These compositions are also effective as adjuvants toenhance the effectiveness of other anti-cancer agents. Such anti-canceragents include, but are not limited to, drugs, immune stimulants,antigens, antibodies, vaccines, radiation, chemotherapeutic agents,genetic, biologically engineered and chemically synthesized agents, andagents that target cell death molecules for activation or inactivation,agents that inhibit proliferation of cancer cells, and that induceapoptosis in cancer cells. MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCare also effective for the prevention or treatment of myelosuppression(monocytopenia and or neutropenia) associated with the treatment ofcancer or cancer by itself, and for the prevention or treatment ofdiverse hematopoietic and myeloid abnormalities associated withmedications or diseases, such as, but not limited to the acquiredimmunodeficiency syndrome (AIDS), myelodysplastic syndromes, autoimmunediseases, end-stage renal diseases or viral infections.

MRNC, MpRNC, MbRNC, MsRNC, MapRNC or MvRNC, in a pharmaceuticallyacceptable carrier, may be administered to an animal or human in adosage effective to stimulate a response in responsive cells of theimmune system, and to inhibit proliferation of and induce apoptosis inresponsive cells. MRNC, MpRNC, MbRNC, MsRNC, MapRNC or MvRNC can beadministered by methods including, but not limited to, suspension inaqueous formulations, in creams and gels, by emulsification in oil orother hydrophobic liquid formulations, enclosure in liposomes, andcomplexation with natural or artificial carriers, with tissue- orcell-specific ligands or with tissue- or cell-specific antibodies.

DEFINITIONS

The term optimization of the manufacturing processes is used to describethe process whereby fermentation to obtain mycobacterial cell massutilizes a cultivation medium where there is an appropriate carbon,nitrogen and iron content resulting in improved yields of mycobacterialcell mass.

The term optimization of the manufacturing process also refers to theelimination of the requirement for the use of chemicals or enzymes inthe manufacture of the mycobacterial cell wall compositions.

Three different names are used in the present application to describethese novel compositions. First, the term bacterial RNA composition(BRNC) is used generally to describe the compositions made fromdifferent species of bacteria. BRNC may be prepared from differentGram-negative or Gram-positive bacteria with the methods disclosedherein. Second, the term mycobacterial RNA composition (MRNC) is used todescribe the compositions made from different mycobacteria. In differentembodiments, MRNC may be prepared from any mycobacterium. Third, inother embodiments, specific mycobacterial species, mycobacterialcomplexes or mycobacterial strains are used to prepare MRNC. Examples ofbut not limited to these are Mycobacterium avium (including sub-speciesparatuberculosis, commonly termed MAP), Mycobacterium bovis BCG,Mycobacterium phlei, Mycobacterium smegmatis, or Mycobacterium vaccaewith the methods disclosed herein. It will be recognized by one of skillin the art that the methods of manufacturing the compositions describedherein are applicable to the manufacture of MRNC from mycobacteria otherthan those described in the detailed description and examples.

The term Mycobacterium RNA composition (MpRNC) is used to describe thecompositions comprising RNA and cell walls made from Mycobacteriumphlei. The terms Mycobacterium bovis strain BCG RNA composition (MbRNC),Mycobacterium smegmatis RNA composition (MsRNC), Mycobacterium aviumsubspecies paratuberculosis (MAP) RNA composition (MapRNC) andMycobacterium vaccae RNA composition (MvRNC) are used to describe thecompositions comprising RNA and cell walls made from Mycobacterium bovisBCG, Mycobacterium smegmatis, Mycobacterium avium subspeciesparatuberculosis and Mycobacterium vaccae, respectively. In eachcomposition, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC, RNA isisolated from mycobacteria using a procedure that results in anefficient production of oligoribonucleotides and polyribonucleotides.

The term mycobacterial RNA composition (MRNC) is used to describe thecompositions made from different mycobacteria. In different embodiments,MRNC may be prepared from any mycobacterium. In other embodiments,specific mycobacterial species, mycobacterial complexes or mycobacterialstrains are used to prepare MRNC. Examples of but not limited to theseare Mycobacterium avium (including sub-species paratuberculosis,commonly termed MAP), Mycobacterium bovis BCG, Mycobacterium phlei,Mycobacterium smegmatis, or Mycobacterium vaccae with the methodsdisclosed herein. It will be recognized by one of skill in the art thatthe methods of manufacturing the compositions described herein areapplicable to the manufacture of cell wall compositions frommycobacteria other than those described in the detailed description andexamples.

The term Mycobacterium phlei RNA composition (MpRNC) is used to describethe compositions made from Mycobacterium phlei. The terms Mycobacteriumbovis strain BCG RNA composition (MbRNC), Mycobacterium smegmatis RNAcomposition (MsRNC), Mycobacterium avium subspecies paratuberculosis(MAP) RNA composition (MapRNC) and Mycobacterium vaccae RNA composition(MvRNC) are used to describe the compositions made from Mycobacteriumbovis BCG, Mycobacterium smegmatis, Mycobacterium avium subspeciesparatuberculosis and Mycobacterium vaccae, respectively. In eachcomposition, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC, RNA isisolated from mycobacteria using a procedure that results in anefficient production of oligoribonucleotides and polyribonucleotides.

As defined herein, Mycobacterial Culture Media Compositions (MCMC) referto novel synthetic cultivation media containing optimized carbon,nitrogen and iron sources that are used to prepare mycobacterial cellmass and where the carbon, nitrogen and iron content of the media aresuch that there is optimal utilization of the carbon and optimalmycobacterial wet cell mass yield.

As defined herein, cultivation refers to the process of generatingmycobacterial cell mass wherein the mycobacteria are cultivated in amedium optimized but not limited to the carbon, nitrogen and ironcontent that ensures optimal division of the bacteria or mycobacteria.Use may be made of equipment and cultivation conditions known to thoseof skill in the art.

As defined herein, down-stream processing or manufacturing refers to theprocess of taking mycobacterial cell mass prepared by cultivation, andmanufacturing RNA-containing compositions or RNA-containing cell wallcompositions by the use of appropriate combinations of high-pressurehomogenization, differential centrifugation and heat treatment. Use maybe made of techniques known to those skilled in the art that arecomparable in effect to high-pressure homogenization without departingfrom the scope or teachings of the invention.

As defined herein, bacterial cell wall refers to a cell wall from amember of the bacterial kingdom that contains cell wall molecularcomponents, and where at a minimum these molecules comprisepolysaccharides and alanine (ALA)-containing peptide chains, commonlycalled peptidoglycan.

As defined herein, mycobacterial cell wall refers to any cell wallcomposition prepared from a member of the family mycobacteriaceae andgenus mycobacterium that contains at a minimum peptidoglycan and mycolicacids, and where the peptidoglycan is composed of a polymer consistingof repeat units of [N-acetylglucosamine-N-acylmuramic acid]_(n) whereN-acyl is either N-acetyl or N-glycolyl, and where the polymeric chainsare linked by peptide bridges, and where the mycolic acids areα-substituted β-hydroxylated very-long-chain fatty acids. The exactamino acid composition of these peptide bridges are highly conservedbetween different mycobacterial species and their strains, and is knownto those skilled in the art, but often includes diaminopimelic acid(DAP) and, as far as is known, always includes alanine (ALA) linked toN-acylmuramic acid through the lactic acid moiety. The types of mycolicacid are specific to individual mycobacterial species, and aresubdivided into clusters depending on the length of the molecule (range˜60-90 carbon atoms).

As defined herein, exogenous contamination refers to the presence of anyexogenous materials, including but not limited to proteins,biochemicals, or chemicals, that are conventionally used in themanufacturing of bacterial- or mycobacterial-derived compositions(including the preparation of bacterial or mycobacterial cell biomassand bacterial or mycobacterial cell wall compositions). In contrast toprevious methods of making bacterial and mycobacterial cell wallcompositions, the bacterial and mycobacterial proteins associated withthe RNC compositions made with the present methods are preserved due tothe absence of exogenous proteolytic enzyme treatments. In contrast toprevious methods of making bacterial and mycobacterial cell wallcompositions, the bacterial and mycobacterial cell wall lipids arepreserved due to the absence of exogenous dilapidating solventtreatments.

As defined herein, the term manufacture refers to the process whereby anRNC is isolated, in which intact bacterial or mycobacterial cells aredisrupted to form cell wall fragments, and where such fragments are thenisolated in combination with RNA to give a bacterial or mycobacterialcell wall RNC.

As defined herein, the terms oligoribonucleotides andpolyribonucleotides refer, respectively, to RNA molecules, of 2 toapproximately 20 bases or of 20 to approximately 4000 bases in lengthobtained from bacteria, mycobacteria, Mycobacterium phlei, Mycobacteriumbovis strain BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis or Mycobacterium vaccae. The RNA togetherwith bacterial or mycobacterial cell walls is defined as RNC. The RNAmay be composed of single strands (ss), double strands (ds),single-double stranded hybrid strands (hs), and the RNA is obtained frombacteria, more preferably from mycobacteria and most preferably fromMycobacterium phlei, Mycobacterium bovis BCG, Mycobacterium smegmatis,Mycobacterium avium subspecies paratuberculosis or Mycobacterium vaccae.The RNC may comprise one or more RNC from different bacteria ormycobacteria, and may additionally comprise intact cells from differentbacteria or mycobacteria. RNC can also be formulated using cell wallsfrom one or more bacteria or mycobacteria.

As defined herein, high-pressure homogenization of bacteria ormycobacteria refers to a process whereby a suspension of intact bacteriaor mycobacteria in an aqueous excipient are passed under pressurethrough a minute gap in a valve creating conditions of high turbulenceand shear force, which cause the disintegration of the bacteria ormycobacteria into cell wall fragments and the release of cytoplasmiccomponents, including protein and lipid.

As defined herein, low-speed centrifugation refers to a relativecentrifugal force (RCF) sufficient to sediment undisrupted bacteria ormycobacteria.

As defined herein, high-speed centrifugation refers to a relativecentrifugal force sufficient to sediment bacterial or mycobacterial cellwall RNC.

As defined herein, immune stimulation refers to the stimulation of theinnate and or acquired immune system, such that an immune response iselicited. This immune response may be comprised of but not limited toone or more of the following: immune cell differentiation and division,activation of Toll-like receptors, activation of NOD receptors,activation of receptors other than Toll-like or NOD, induction ofchemokine and cytokine synthesis, induction of growth factor synthesis,increased expression of cell surface markers, decreased expression ofcell surface markers, activation of cytocidal or cytotoxic activity,stimulation of antibody production or stimulation of cell-mediatedimmunity.

As defined herein, anticancer activity refers to any process thatresults in the inhibition of and or death of cancer cells. Suchanticancer activity may be the result of a direct effect on cancer celltargets, or the result of an indirect effect due to stimulation of theimmune system.

The term animal includes human in this application.

The term produce means synthesize and/or release in this application.

Novel Compositions for the Cultivation of Mycobacteria

The present invention provides novel media for cultivating mycobacteriathat contain optimized levels and ratios of carbon, nitrogen and ironfor the generation of mycobacterial cell mass in a timely manner. Thesenovel media utilize organic nitrogen sources or inorganic nitrogensources, and provide for procedures where additional carbon, nitrogenand iron may be provided within one molecule. These novel cultivationmedia do not require the addition of exogenous proteins such as bovinealbumin or catalase for the efficient production of mycobacterial cellmass.

In one embodiment, the optimized nitrogen is provided by the addition ofinorganic ammonium salts including but not limited to ammonium sulfate.The optimized carbon is provided by the addition of organic moleculesincluding but not limited to sugars such as glucose (dextrose), glycolssuch as glycerol or acids such as citric acid, and that contain carbonthat can be metabolized by the mycobacteria.

In another embodiment, the optimized nitrogen and carbon are provided bymolecules containing both atoms including but not limited to ammoniumcitrate (dibasic) or amino acids such as but not limited to asparagine.

In yet another embodiment the optimized carbon, nitrogen and iron areprovided by molecules containing all three atoms including but notlimited to ferric ammonium citrate.

In a further embodiment, use may be made of cultivation media thatprovide optimized carbon, nitrogen and iron as well as additionaldivalent cations including but not limited to calcium and magnesium.

In yet another embodiment, the cultivation media are formulated withoutadditional exogenous biological agents including but not limited toalbumins, enzymes or growth enhancing materials including but notlimited to mycobactin.

In yet another embodiment, use is made of mixing and aeration proceduresto optimize access to oxygen to ensure rapid aerobic growth of thebacteria or mycobacteria

Novel Bacterial and Mycobacterial Compositions

The present invention provides novel bacterial and mycobacterialcompositions comprise RNA isolated from bacteria or mycobacteria, abacterial or mycobacterial cell wall. These compositions include BRNC,MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC, as previously defined.These compositions optionally contain different amounts of intactbacterial or mycobacterial cells. In another embodiment, eachcomposition, MRNC, MpRNC, MbRNC, MsRNC, MapRNC or MvRNC, may alsocontain different amounts of intact mycobacterial, Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis, or Mycobacterium vaccae cells. Intact cellsmay be present in MRNC, MpRNC, MbRNC MsRNC, MapRNC or MvRNC in amountsof from 0-99%, 0.05-95%, 0.07-50%, 0.1-20% or 0.19-19% by weight, or inan amount within any of these ranges.

In another embodiment, these novel compositions possess an RNAcomposition content (which can range from approximately 30% to 100% ofthe total nucleic acid content) in the form of oligoribonucleotides andpolyribonucleotides of between 2 and 4000) bases in length. In yetanother embodiment, these novel compositions possess an RNA content(30-100% of the total nucleic acid content) in the form ofoligoribonucleotides and polyribonucleotides that are greater than 150bases, or of between 151 to 4000 bases in length. In another embodiment,these novel compositions possess an RNA content (30-100% of the totalnucleic acid content) that is mostly in the form of oligoribonucleotidesand polyribonucleotides that are between 20 and 40 bases in length.

In one embodiment, novel RNC compositions containing about 90% or moreRNA in the form of oligoribonucleotides and polyribonucleotides areprepared by extraction of RNA from mycobacteria, Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis (MAP) or Mycobacterium vaccae, using forexample guanidinium thiocyanate-phenol-chloroform extraction. Such RNApreparations may be heat-treated (by for example but not limited to 121°C. for between 5-30 min) in order to give an RNC composition where theoligoribonucleotides and polyribonucleotides chain length is between 2and 4000 bases. This RNC composition may be combined with apharmaceutically acceptable carrier.

Anti-cancer activity is optimized when there is minimal content ofintact mycobacterial cells. Immune stimulatory activity is optimizedwhen there is a content of intact mycobacterial cells in the range of5-50% w/w. The number of intact bacterial, mycobacterial, Mycobacteriumphlei, Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacteriumavium subspecies paratuberculosis (MAP) or Mycobacterium vaccae cellscan be modulated by controlling the manufacturing process as detailed inthe examples, or by adding intact mycobacterial, Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis (MAP) or Mycobacterium vaccae cells to MRNC,MpRNC, MbRNC, MsRNC, MapRNC, or MvRNC.

These novel compositions, MRNC, MpRNC, MbRNC, MsRNC, MapRNC, or MvRNC,possess immune stimulating, anti-cancer and hematopoietic and myeloidcell growth factor stimulating activity. As opposed to prior artpreparations, these new compositions contain single, single strands(ss), double strands (ds) and single-double stranded hybrid strands(hs), are optimized for immune stimulatory and anticancer activity, andare formulated with mycobacterial, Mycobacterium phlei, Mycobacteriumbovis BCG, Mycobacterium smegmatis, Mycobacterium avium subspeciesparatuberculosis (MAP) or Mycobacterium vaccae cell walls. In oneembodiment these compositions further comprise levels of intactmycobacterial cells appropriate for the intended application.

The compositions of the present invention are effective in stimulatingthe immune system, in stimulating hematopoietic and myeloid growthfactor synthesis, and in treating cancer. In different embodiments,these compositions are effective in inducing a response in responsivecells of an animal's immune system, in inducing cell cycle arrest orapoptosis and inhibiting cellular proliferation. These compositionsinduce responsive cells of the immune system to produce cytokines,chemokines and hematopoietic and myeloid growth factors.

The compositions of the present invention are effective in stimulatingthe immune system and in treating cancer when combined with apharmaceutically acceptable carrier to form a therapeutic compositionand administered to an animal. In different embodiments, thesetherapeutic compositions induce a response in responsive cells of ananimal's immune system, induce cell cycle arrest or apoptosis andinhibit cancer cell proliferation. These compositions induce responsivecells of the immune system to produce cytokines, chemokines andhematopoietic and myeloid cell growth factors.

MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are effective therapeuticagents in preventing, treating, lessening and eliminating a variety ofdiseases including, but not limited to, malignant, autoimmune andimmunodeficiency diseases, myelosuppression and hematopoietic andmyeloid abnormalities. MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC areparticularly useful for treating diseases and processes mediated byundesired and uncontrolled cell proliferation, such as but not limitedto cancer. These compositions are also effective as adjuvants to enhancethe effectiveness of other anti-cancer agents. Such anti-cancer agentsinclude, but are not limited to, drugs, immune function stimulants,immune function inhibitors, antigens, antibodies, vaccines, radiation,chemotherapeutic agents (either alone or in appropriate combinationsknown to those of skill in the art), genetic, biologically engineeredand chemically synthesized agents, and agents that target cell deathmolecules for activation or inactivation, agents that inhibitproliferation of cancer cells, and that induce apoptosis in cancercells. MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are also effectivefor the prevention or treatment of myelosuppression (leucopenia,neutropenia,) associated with the treatment of cancer or induced by thecancer itself, and for the prevention or treatment of diversehematopoietic and myeloid abnormalities associated with medications ordiseases, such as, but not limited to the acquired immunodeficiencysyndrome (AIDS), myelodysplastic syndromes, autoimmune diseases,end-stage renal diseases or viral infections. Combinations of two ormore compositions of the present invention may be used to optimizemodulation of the immune system and to treat cancer. For example, acombination of MpRNC and MvRNC will give optimal NOD2 and TLR2activation for immune stimulation and cytokine induction. Similarly, acombination of MpRNC and MvRNC will give optimal anticancer activity andimmune stimulation.

In one embodiment, bacteria may be used to provide a BRNC compositionthat comprises bacterial RNA in the form of oligoribonucleotides andpolyribonucleotides, bacterial cell walls, and a pharmaceutical carrier,excipient or vehicle. In another embodiment mycobacteria may be used toprovide an MRNC composition comprising mycobacterial RNA in the form ofoligoribonucleotides and polyribonucleotides, mycobacterial cell walls,and a pharmaceutical carrier or vehicle. In another embodiment theMpRNC, MbRNC, MsRNC, MapRNC or MvRNC compositions comprise Mycobacteriumphlei RNA, Mycobacterium bovis BCG RNA, Mycobacterium smegmatis RNA,Mycobacterium avium subspecies paratuberculosis RNA or Mycobacteriumvaccae RNA respectively, in the form of oligoribonucleotides andpolyribonucleotides, in combination with cell walls from these species,and a pharmaceutical carrier, excipient or vehicle.

In one embodiment the BRNC, MRNC, MpRNC MbRNC, MsRNC or MvRNCcompositions are formulated with a pharmaceutical delivery system suchas but not limited to chitosan nanoparticles or cationic liposomes.

Each of these compositions, BRNC, MRNAC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC may be combined with an acceptable carrier, such as apharmaceutically acceptable carrier, excipient, vehicle or otherdelivery system known to those of skill in the art to form a compositionthat may be administered to an animal or a human. Each of thesecompositions, BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC may becombined with selected amounts of intact bacterial cells, mycobacterialcells, Mycobacterium phlei, Mycobacterium bovis BCG, Mycobacteriumsmegmatis, Mycobacterium avium subspecies paratuberculosis orMycobacterium vaccae cells, respectively, to form a composition that maybe administered to an animal or a human.

BRNC, MRNAC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are effective ininducing responsive cells of the immune system to produce cytokines,chemokines and hematopoietic and myeloid growth factors, and ininhibiting proliferation of and inducing apoptosis in responsive cellsincluding, but not limited, to cancer cells, in an animal.

Eubacterial species can be used to practice the present inventionincluding, but not limited to, Coryneform species, Corynebacteriumspecies, Rhodococcus species, Bordetella species, Escherichia species,Listeria species, Nocardia species and Mycobacterial species to makeBRNC or MRNC. Mycobacterial species including, but not limited to,Mycobacterium smegmatis, Mycobacterium fortuitum, Mycobacterium kansaii,Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium vaccae,Mycobacterium avium and associated subspecies and Mycobacterium phleican be used to make MRNC. In yet another embodiment, the mycobacteriumspecies Mycobacterium phlei, Mycobacterium bovis BCG, Mycobacteriumsmegmatis, Mycobacterium avium subsp. paratuberculosis or Mycobacteriumvaccae are used to make MpRNC, MbRNC, MsRNC, MapRNC or MvRNC. In yetanother embodiment Archaebacterial species can be used to prepare BRNC.

Method for Manufacturing Mycobacterial Cell Wall RNA ContainingCompositions (MRNC)

The present invention additionally provides a method for manufacturingmycobacterial cell wall RNA compositions from any mycobacterial species.Comprehensive listings of mycobacterial species, mycobacterialcomplexes, mycobacterial sub-species and mycobacterial strains that canbe used to prepare mycobacterial cell wall-RNA using the method of thepresent invention are maintained by and readily available from forexample the NCBI of the USA. Although the present invention providesmethods for the manufacture of MRNC from all mycobacterial species andstrains, preferred mycobacterial species and strains are those that areknown to be fast-growing in culture, and thus capable of providingmycobacterial cell biomass in short periods of time. Also preferred arethose mycobacterial species and strains that are known to befast-growing and are additionally non-pathogenic to immunocompetentindividuals.

BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are relativelyinexpensive to prepare, their activity is reproducible amongpreparations and remains stable over time, and they are not contaminatedby exogenous materials, including but not limited to proteins, enzymes,biochemicals r chemicals used by those of ordinary skill in the art toprepare mycobacterial cell wall compositions. Further, the BRNC, MRNC,MpRNC, MbRNC, MsRNC, MapRNC and MvRNC preparations are minimally, if atall, toxic to the recipient

To prepare BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC, bacterialspecies, mycobacterial species, Mycobacterium phlei, Mycobacterium bovisBCG, Mycobacterium smegmatis, Mycobacterium avium subspeciesparatuberculosis or Mycobacterium vaccae respectively, are grown inappropriate liquid media with an optimized carbon, nitrogen and ironcontent that assures complete utilization of carbon sources, and areharvested. Amino acids provide a source of nitrogen. Dibasic amino acidsincluding but not limited to asparagine are preferred sources ofnitrogen. Other sources of nitrogen include ammonium salts or theirequivalent. Additional sources of iron are inorganic salts, or organicsalts, including but not limited to citrate, that provide additionalsources of carbon. Iron complexes, including but not limited to ferricammonium citrate (ammonium ferric citrate), provide additional iron,nitrogen and carbon sources. Preferred concentrations of carbon (asglucose or other metabolically available carbon source) are 500-2500mMol/liter. In different embodiments, ratios of C to N are from about1:0.0095 to 1:0.06, from about 1:0.02 to 1:0.06, or at or about 1:0.06.In different embodiments, ratios of C to Fe are at or about 1:1.8×10⁻⁴to 1:3.2×10⁻³; at or about 1:1.8×10⁻³ to 2.7×10⁻⁴; or at or about1:2.7×10⁻⁴. Utilization may be made of any acceptable source of carbon,nitrogen or iron known to those of skill in the art. Such media may alsocontain additional salts as well as vitamins, and it is to be expectedthat the presence and concentrations of these will be adjusted accordingto the particular needs.

The bacteria or mycobacteria are disrupted to liberate RNA and to ensurethe controlled removal of intact bacteria, mycobacteria, Mycobacteriumphlei, Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacteriumavium subspecies paratuberculosis or Mycobacterium vaccae cells.Disruption occurs via the use of a high-pressure homogenization processfollowed by a centrifugation process. Low relative centrifugal forceremoves intact bacteria or mycobacteria. RNA from BRNC, MRNC, MpRNC,MbRNC, MsRNC, MapRNC and MvRNC may be extracted at this stage usingguanidinium thiocyanate-phenol-chloroform or similar procedures known tothose of skill in the art. Alternatively, high relative centrifugalforces can be used to isolate BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNCand MvRNC formulated with bacterial, mycobacterial, Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis or Mycobacterium vaccae cell walls afterbacterial or mycobacterial cellular disruption. Importantly, nuclease-,and DNase/RNase-free (both endo- and exo-nuclease activity) reagents areused and the process is carried out at or about 4° C. to minimize RNAdegradation during the preparation steps.

The oligoribonucleotide and polyribonucleotide sequences,oligoribonucleotide and polyribonucleotide length, andoligoribonucleotide and polyribonucleotide structures (such as but notlimited to single stranded molecules, double stranded molecules, hybridscontaining single and double strands or intra-oligoribonucleotide andintra-polyribonucleotide base-pairing structures) of bacterial RNA arenecessary for the biological activity of BRNC. More specifically, theoligoribonucleotide and polyribonucleotide sequences,oligoribonucleotide and polyribonucleotide length, andoligoribonucleotide and polyribonucleotide structures (such as but notlimited to single stranded molecules, double stranded molecules, hybridscontaining single and double strands or intra-oligoribonucleotide andintra-polyribonucleotide base-pairing structures) of mycobacterial RNAare necessary for biological activity of MRNC. The use of bacterial,mycobacterial or Mycobacterium phlei, Mycobacterium bovis BCG,Mycobacterium. smegmatis, Mycobacterium avium subsp. paratuberculosis orMycobacterium vaccae to formulate BRNC, MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC cell walls results in a biological carrier and deliverysystem that is important for maximizing the biological activity (immunestimulation and anti-cancer activity) of BRNC, MRNC, MpRNC, MbRNC,MsRNC, MapRNC and MvRNC.

Methods of Making BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCContaining Different Numbers of Intact Bacterial or Mycobacterial Cells

The present invention additionally provides a method for manufacturingBRNC from any bacterial species and strain. Additionally, the presentinvention additionally provides a method for manufacturing MRNC from anymycobacterial species. Comprehensive listings of mycobacterial species,mycobacterial complexes, mycobacterial sub-species and mycobacterialstrains that can be used to prepare mycobacterial cell wall-RNC usingthe method of the present invention are maintained by and readilyavailable from for example the NCBI of the USA. Preferred mycobacterialspecies are those that are known to be fast-growing in culture, and thuscapable of providing mycobacterial biomass in short periods of time.Also preferred are species that are known to be fast-growing andrecognized as being non-pathogenic to immunocompetent individuals.

In one embodiment, the number of intact bacterial cells present in BRNC,MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC can be controlled by theappropriate use of a defined high-pressure during homogenization and inconjunction with a defined number of homogenization cycles that are usedin the preparation of BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCformulations containing bacterial, mycobacterial or Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis or Mycobacterium vaccae cell walls. Theinventors have unexpectedly found that varying the number of intactbacterial cells present in BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC can affect the immune stimulatory and anti-cancer activity ofthese compositions such that the desired and optimal immune modulatingor desired and optimal anti-cancer activity can be achieved.

In one embodiment, the number of intact mycobacterial cells inmycobacterial, Mycobacterium phlei, Mycobacterium bovis BCG,Mycobacterium smegmatis, Mycobacterium avium subspecies paratuberculosisor Mycobacterium vaccae cell wall formulations of MRNC, MpRNC, MbRNC,MsRNC, MapRNC and MvRNC can be reduced at least 20-fold compared to anequivalent sample of mycobacteria that has not undergone high-pressurehomogenization and low-speed centrifugation treatment. In anotherembodiment, the number of intact mycobacterial cells in, MpRNC, MbRNC,MsRNC, MapRNC and MvRNC can be reduced at least 30-fold compared to anequivalent sample of mycobacteria, Mycobacterium phlei, Mycobacteriumbovis BCG, Mycobacterium smegmatis, Mycobacterium avium subspeciesparatuberculosis or Mycobacterium vaccae that has not undergonehigh-pressure homogenization, low-speed centrifugation followed byadditional high-pressure homogenization treatment of the bacterial cellpreparation. In a further embodiment, the number of intactmycobacterial, Mycobacterium phlei, Mycobacterium bovis BCG,Mycobacterium smegmatis, Mycobacterium avium subspecies paratuberculosisor Mycobacterium vaccae cells in MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC can be reduced at least 35-fold compared to an equivalent sampleof the mycobacteria that has not undergone high-pressure homogenization,low-speed centrifugation followed by additional high-pressurehomogenization treatment of the mycobacterial or Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis or Mycobacterium vaccae cell preparation.

In one embodiment, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCcompositions containing high levels of intact mycobacterial cell contentor Mycobacterium phlei, Mycobacterium bovis BCG, Mycobacteriumsmegmatis, Mycobacterium avium subspecies paratuberculosis orMycobacterium vaccae cell content, respectively, (e.g. aboveapproximately 1-50% w/w) may be prepared. Such techniques include forexample four cycles of high-pressure homogenization, wherein two of thefour cycles of high-pressure homogenization are conducted before ahigh-speed centrifugation step, and two of the high-pressurehomogenization steps are conducted after the high-speed centrifugationstep.

In another embodiment, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCpreparations containing intermediate levels of intact mycobacterial cellcontent, Mycobacterium phlei, Mycobacterium bovis BCG, Mycobacteriumsmegmatis, Mycobacterium avium subspecies paratuberculosis orMycobacterium vaccae cell content, respectively, (e.g. between fromabout 0.2 to about 0.9% w/w) may be prepared. Such techniques include,for example seven cycles of high-pressure homogenization, wherein fiveof the seven cycles of high-pressure homogenization are conducted beforea high-speed centrifugation step, and two high-pressure homogenizationsteps are conducted after the high-speed centrifugation step.

In a further embodiment, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCpreparations containing ultra-low levels of intact mycobacterial cellcontent, Mycobacterium phlei, Mycobacterium bovis BCG, Mycobacteriumsmegmatis, Mycobacterium avium subspecies paratuberculosis orMycobacterium vaccae cell content (e.g. less than from about 0.2% w/w)may be prepared. Such techniques include for example ten cycles ofhigh-pressure homogenization, wherein five of the ten cycles ofhigh-pressure homogenization are conducted before a low-speedcentrifugation step, three cycles of high-pressure homogenization areconducted after the low-speed centrifugation and two high-pressurehomogenization steps are conducted after the high-speed centrifugationstep.

In the above techniques, ‘high-pressure homogenization’ means a cycle ofhigh-pressure homogenization sufficient to cause the efficient ruptureof mycobacteria, Mycobacterium phlei, Mycobacterium bovis BCG,Mycobacterium smegmatis, Mycobacterium avium subspecies paratuberculosisor Mycobacterium vaccae such that cell wall fragment compositionscontaining RNA are obtained. Other procedures that are comparable tohigh-pressure homogenization such as, but not limited to,microfluidization can also be used. It is to be understood that one ofordinary skill in the art of bacterial and mycobacterial celldisruption, after reading the present invention, can readily determinethe optimal pressure for the specific mycobacterial species, strain,substrain, or complex that is to be disrupted.

As defined herein, low-speed centrifugation refers to a relativecentrifugal force (RCF) sufficient to sediment undisrupted bacteria,mycobacteria, Mycobacterium phlei, Mycobacterium bovis BCG,Mycobacterium smegmatis, Mycobacterium avium subspecies paratuberculosisor Mycobacterium vaccae. It is to be understood that one of ordinaryskill in the art of centrifugation, after reading the present invention,can readily determine the optimal RCF for sedimentation of any intactbacteria or mycobacteria that remain following cell disruption asdescribed above.

High-speed centrifugation, as defined herein, refers to a relativecentrifugal force sufficient to sediment bacterial, mycobacterial,Mycobacterium phlei, Mycobacterium bovis BCG, Mycobacterium smegmatis,Mycobacterium avium subspecies paratuberculosis or Mycobacterium vaccaecell walls. It is to be expected that one of ordinary skill in the artof centrifugation, after reading the present invention, can readilydetermine the optimal relative centrifugal force for the sedimentationof cell walls from the bacterium or mycobacterium following disruptionand removal of the desired amount of intact bacteria and mycobacteria bylow speed centrifugation as described above.

The use of the above high-pressure homogenization techniques can be usedto prepare MRNC containing different levels of intact mycobacteria (forexample, of high, intermediate or low) by the appropriate use of highpressure homogenization pressures, low speed centrifugation andhigh-speed centrifugation, or combinations thereof. Additionally, thehigh-pressure homogenization techniques described have been used toprepare MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC containing high, lowor intermediate levels of mycobacteria, Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis or Mycobacterium vaccae, respectively.

In one embodiment, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCpreparations, prepared via the high-pressure homogenization methodsdescribed above, contain reduced numbers of intact mycobacterial cells,Mycobacterium phlei, Mycobacterium bovis BCG, Mycobacterium smegmatis,Mycobacterium avium subspecies paratuberculosis or Mycobacterium vaccaecells, respectively, as compared to mycobacterial preparations that haveundergone differential centrifugation to remove intact mycobacterialcells, Mycobacterium phlei, Mycobacterium bovis BCG, Mycobacteriumsmegmatis, Mycobacterium avium subspecies paratuberculosis orMycobacterium vaccae cells from the preparation (i.e., no high pressurehomogenization treatment). In one embodiment, the percent of intactmycobacterial cells, Mycobacterium phlei, Mycobacterium bovis BCG,Mycobacterium smegmatis, Mycobacterium avium subspecies paratuberculosisor Mycobacterium vaccae cells per MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC preparation (w/w), respectively, is less than about 50% w/w, lessthan about 40% w/w, less than about 30% w/w, less than about 20% w/w,less than about 10% w/w, less than about 5% w/w, less than about 1% w/w,or less than about 0.5% w/w.

In another embodiment, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCpreparations are subjected to a process of heat treatment such as butnot limited to, for example, heating at 95° C. for 5-30, min or forexample heating at 121° C. for 5-30 min, that is sufficient toinactivate any intact mycobacteria, Mycobacterium phlei, Mycobacteriumbovis BCG, Mycobacterium smegmatis, Mycobacterium avium subspeciesparatuberculosis or Mycobacterium vaccae remaining in the composition.

In another embodiment, heat treatment (for example 121° C., 5-30 min orother conditions known to those of skill in the art) of MRNC, MpRNC,MbRNC, MsRNC, MapRNC and MvRNC preparations is used to generateoligoribonucleotides and polyribonucleotides in the range of about 2 to150, of about 10 to 100, or about 20 to 40 bases in length.

In one embodiment, a method of making MRNC, MpRNC, MbRNC, MsRNC, MapRNCand MvRNC comprises disrupting a mycobacterial cell biomass,Mycobacterium phlei, Mycobacterium bovis BCG, Mycobacterium smegmatis,Mycobacterium avium subspecies paratuberculosis or Mycobacterium vaccaeto prepare cell walls there from and associated oligoribonucleotides andpolyribonucleotides, separating intact non-disrupted mycobacterial,Mycobacterium phlei, Mycobacterium bovis BCG, Mycobacterium smegmatis,Mycobacterium avium subspecies paratuberculosis or Mycobacterium vaccaecells; and separating the soluble, cytosolic contents of the portion ofthe disrupted bacterial mycobacterial, Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis or Mycobacterium vaccae cells from the cellwalls and oligoribonucleotides and polyribonucleotides to obtain abacterial, mycobacterial, Mycobacterium phlei, Mycobacterium bovis BCG,Mycobacterium smegmatis, Mycobacterium avium subspecies paratuberculosisor Mycobacterium vaccae cell wall RNC (BRNC, MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC).

In another embodiment, a method of making MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC preparations comprises disrupting mycobacterial cellbiomass to release oligoribonucleotides and polyribonucleotides;separating the oligoribonucleotides and polyribonucleotides from thenon-disrupted, intact mycobacterial, Mycobacterium phlei, Mycobacteriumbovis BCG, Mycobacterium smegmatis, Mycobacterium avium subspeciesparatuberculosis or Mycobacterium vaccae cells; repeating the steps tocontrol the number of intact mycobacterial, Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis or Mycobacterium vaccae cells; and, thenheating the oligoribonucleotides and polyribonucleotides to atemperature sufficient to obtain an oligoribonucleotide andpolyribonucleotide length of about 20 to 40 nucleotide bases.

Nucleic Acid Content of the Compositions

The nucleic acid content of these compositions, MRNC, MpRNC, MbRNC,MsRNC, MapRNC and MvRNC, is from about 500 to 50000 ng/mg, from about500 to 5000 ng/mg or from about 500 to 3000 ng/mg. In differentembodiments, the length of extracted oligoribonucleotides andpolyribonucleotides of MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC isabout 2 to about 4000 bases in length, about 2 to about 150 bases inlength, or greater than 150 bases in length. In different embodiments,the length of extracted oligoribonucleotides and polyribonucleotides ofMRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC is about 150 bases to about4000 bases in length, or about 10 to about 50 bases in length, or about20 to about 40 bases in length. In different embodiments, the length ofthe extracted oligoribonucleotides and polyribonucleotides of MRNC,MpRNC, MbRNC, MsRNC, MapRNC and MvRNC is less than 150 bases, or lessthan 100 bases.

It will be apparent to one of ordinary skill in the art, that the aboveprocedures may be modified such that other steps are included, providingthey do not negatively impact the quality and overall recovery of MRNC,MpRNC, MbRNC, MsRNC, MapRNC and MvRNC.

MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC increase in theirtherapeutic effectiveness upon high-pressure homogenization andlow-speed centrifugation, because the high-pressure homogenizationprocedure reduces the length of the extracted RNA molecules bygenerating oligoribonucleotides and polyribonucleotides, and removesintact mycobacterial, Mycobacterium phlei, Mycobacterium bovis BCG,Mycobacterium smegmatis, Mycobacterium avium subspecies paratuberculosisor Mycobacterium vaccae cells from the respective preparation. Furtherreductions in the oligoribonucleotide and polyribonucleotide length canbe readily achieved by the use of, but not limited to heat treatment(for example, by heat treatment at 121° C. for 30 min) or by theappropriate use of base-sequence restricted endonucleases. The inventorshave unexpectedly discovered that MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC preparations are significantly reduced in their ability to act asimmune stimulants or anticancer agents after RNase treatment, whichdigests RNA. Accordingly, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC,preparations have limited therapeutic effectiveness upon RNase treatmentor inadvertent exposure to ribonucleases unless the RNA is protected bythe use of formulations and carrier systems that restrict access of theRNA to the degradative action of RNase (endo or exo) or nucleases.

Immune Stimulatory and Anticancer Activity of MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC

Reduction in the total number of intact mycobacterial, Mycobacteriumphlei, Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacteriumavium subspecies paratuberculosis or Mycobacterium vaccae cells presentin MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC preparations were foundto have a significant effect on immune stimulatory activity as measuredby cytokine induction assays. A reduction in the total number of intactmycobacterial cells present in MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC was also found to have a significant positive effect on immunestimulatory activity, such as increased IL-10 and IL-12p40 induction inperipheral blood mononuclear cells. Furthermore, a reduction in thetotal number of intact mycobacterial, Mycobacterium phlei, Mycobacteriumbovis BCG, Mycobacterium smegmatis, Mycobacterium avium subspeciesparatuberculosis or Mycobacterium vaccae cells present in the abovepreparations was found to have a positive, enhancing effect on theanticancer activity of MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC, asmeasured by the inhibition of cancer cell proliferation.

In one embodiment, the immune stimulatory activity of MRNC, MpRNC,MbRNC, MsRNC, MapRNC and MvRNC on hematopoietic and myeloid growthfactors in an animal or human can be measured using cytokine inductionassays. In another embodiment, MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC induce immune stimulatory activity of cytokines, including but notlimited to, IL-10 and IL-12.

In a further embodiment, therapeutic compositions comprising MRNC,MpRNC, MbRNC, MsRNC, MapRNC and MvRNC possess anti-cancer activity. Inone embodiment, the anti-cancer activity of MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC C may be determined using cancer cell proliferationassays known to those of ordinary skill in the art. The use of suchassays to identify anticancer activity is known to those of ordinaryskill in the art to be predictive of in vivo anticancer activity.

Administration of compositions comprising MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC in a prophylactic or therapeutic setting is not initself an immunization process. It is a prophylactic or therapeutictreatment that stimulates a response in responsive cells of the immunesystem, and that inhibits proliferation of, and induces apoptosis inresponsive cells such as but not limited to cancer cells. Thisprophylactic or therapeutic treatment is useful to prevent, treat, abateor eliminate a disease including, but not limited to, cancer.

It is to be recognized however that administration of MRNC, MpRNC,MbRNC, MsRNC, MapRNC and MvRNC preparations will elicit an immuneresponse against mycobacterial cell wall components when used in theformulation, that is additionally enhanced by the immune stimulatoryactivity of the MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC (immuneadjuvant effect). Those of ordinary skill in the art will recognize thatsuch a response will be effective in treating opportunistic orpathogenic mycobacterial infections when the MRNC is prepared therefrom. Specifically, there is a need for a treatment that is capable ofreducing the inflammation and morbidity associated with but not limitedto tuberculosis, Johne's disease or Crohn's disease. More specificallythe use of compositions comprising MapRNC with optimized immunestimulatory and vaccine antigen and vaccine adjuvant activity may beused with advantage to prevent or treat MAP infections in cattle andhumans. Compositions comprising RNC from more than one mycobacterialspecies that contain antigens that cross-react with those of MAP may beused to give rise to a protective response that results in control ofMAP infection.

The therapeutic effects of compositions comprising MRNC, MpRNC, MbRNC,MsRNC, MapRNC and MvRNC include, but are not limited to, stimulation ofresponsive cells of the immune system to produce cytokines, which canresult in activation of immune system cells and subsequent cytolysis, oractivation of caspases and apoptosis, in responsive cells. Cytolysis andapoptosis, both individually and in combination, have both anticanceractivity and adjuvant activity. That is, therapeutic compositionscomprising MRNC, MpRNC, MbRNC, MsRNC, and MvRNC can be administeredalone as an anti-cancer agent and that compositions comprising MRNC,MpRNC, MbRNC, MsRNC, MapRNC and MvRNC can be administered before, at thesame time as, or after another anti-cancer agent to increase treatmenteffectiveness.

MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are effective prophylacticand therapeutic agents in preventing, treating, lessening andeliminating a variety of diseases including, but not limited to,malignant, autoimmune and immunodeficiency diseases, myelosuppressionand hematopoietic and myeloid abnormalities. MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC are also effective as adjuvants to enhance theeffectiveness of other agents. Such agents include, but are not limitedto, drugs, antibiotics, other immune stimulants, antigens, antibodies,vaccines, radiation and chemotherapeutic, genetic, biologicallyengineered and chemically synthesized agents, and agents that targetcell death molecules for activation or inactivation, that inhibitproliferation of, and that induce apoptosis in cancer cells.

In each of the aforementioned aspects and embodiments of the invention,combination adjuvants or therapies other than those described above arealso specifically contemplated herein.

In one embodiment, the compositions of the present invention may beadministered with one or more of currently available chemotherapeuticagents known to one of ordinary skill in the art.

In another embodiment, the compositions of the present invention may beadministered with one or more antibiotics, anti-fungicides, anti-viralagents, anti-parasitic agents, anti-inflammatory or immune stimulatorydrugs or agents known to one of ordinary skill in the art.

In yet another embodiment, the compositions of the present invention maybe administered with one or more drugs or agents known to one ordinaryskill in the art for the prevention or treatment of myelosuppression orhematopoietic and myeloid abnormalities.

The compositions of the instant invention may be used for the treatmentof animal subjects or patients, and more preferably, mammals, includinghumans, as well as mammals such as non-human primates, dogs, cats,horses, cattle, swine, rodents and fish.

Pharmaceutically Acceptable Carriers and Methods of Administering theCompositions

The composition of MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC may bereadily formulated with, prepared with, or administered with, apharmaceutically acceptable carrier. Such preparations may be preparedby various techniques. Such techniques include bringing into associationthe composition of MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC and itscarrier. In one embodiment, the MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC compositions are prepared by uniformly and intimately bringinginto association the MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCcompositions with liquid carriers, with solid carriers, or with both.Liquid carriers include, but are not limited to, aqueous formulations,non-aqueous formulations, or both. Solid carriers include, but are notlimited to, biological carriers, chemical carriers, or both.

MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC compositions may beadministered in an aqueous suspension, an oil emulsion, water in oilemulsion and water-in-oil-in-water emulsion, and in carriers including,but not limited to, creams, gels, liposomes (neutral, anionic orcationic), lipid nanospheres or microspheres, neutral, anionic orcationic polymeric nanoparticles or microparticles, site-specificemulsions, long-residence emulsions, sticky-emulsions, micro-emulsions,nano-emulsions, microspheres, nanospheres, nanoparticles and minipumps,and with various natural or synthetic polymers that allow for sustainedrelease of the composition including anionic, neutral or cationicpolysaccharides and anionic, neutral cationic polymers or copolymers,the minipumps or polymers being implanted in the vicinity of wherecomposition delivery is required. Polymers and their use are describedin, for example, Brem et al. (Journal of Neurosurgery 1991, 74:441-446).Furthermore, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC can be usedwith any one, or any combination of, carriers. These include, but arenot limited to, anti-oxidants, buffers, and bacteriostatic agents, andmay include suspending agents and thickening agents.

In one embodiment, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCcompositions or carrier-based preparations are administered as anRNase-free, aqueous suspension. For administration as an aqueoussuspension MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are suspended ina RNase-free and nuclease free pharmaceutically acceptable excipient,buffer or carrier including, but not limited to, water for injection,physiological saline or dextrose solutions, or by techniques including,but not limited to, high-pressure homogenization, sonication andmicrofluidization, and can be aseptically processed or terminallysterilized. In another example, freeze-dried (lyophilized) MRNC, MpRNC,MbRNC, MsRNC, MapRNC and MvRNC can be stored in sealed ampoules or vialsrequiring only the addition of a pharmaceutically acceptable excipient,buffer or carrier, for example RNase-free and nuclease-free sterilewater, immediately prior to use.

For administration in a non-aqueous carrier, MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC may be emulsified with a mineral oil or with a neutraloil such as, but not limited to, a diglyceride, a triglyceride, aphospholipid, a lipid, an oil and mixtures thereof, wherein the oilcontains an appropriate mix of polyunsaturated and saturated fattyacids. Examples include, but are not limited to, soybean oil, canolaoil, palm oil, olive oil and myglyol, wherein the number of fatty acidcarbons is between 12 and 22 and wherein the fatty acids can besaturated or unsaturated. Optionally, charged lipid or phospholipid canbe suspended in the neutral oil. More specifically, use can be made ofphosphatidylserine, which targets receptors on macrophages. Use can bemade of MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC formulated inaqueous media or of lyophilized MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC powder in preparing emulsions using techniques known to those ofordinary skill in the art.

The invention thus provides MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCcompositions together with one or more pharmaceutically acceptablecarriers and, optionally, other therapeutic and/or prophylacticingredients. The carrier and other therapeutic ingredients must beacceptable in the sense of being compatible with the other ingredientsof the composition and not deleterious to the recipient thereof

The MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC compositions areadministered in an amount effective to induce a therapeutic response inan animal, including a human. The dosage of MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC composition administered will depend on the conditionbeing treated, the particular formulation, and other clinical factorssuch as weight and condition of the recipient and route ofadministration. In one embodiment, the amount of MRNC, MpRNC, MbRNC,MsRNC, MapRNC and MvRNC administered is from about 0.00001 mg/kg toabout 100 mg/kg per dose. In another embodiment, the amount of MRNC,MpRNC, MbRNC, MsRNC, MapRNC and MvRNC administered is from about 0.0001mg/kg to about 50 mg/kg per dose. In a further embodiment, the amount ofMRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC administered is from about0.001 mg/kg to about 10 mg/kg per dose. In another embodiment, theamount of MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC administered isfrom about 0.01 mg/kg to about 5 mg/kg per dose. In a furtherembodiment, the amount of MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCadministered is from about 0.1 mg/kg to about 1 mg/kg per dose.

Useful dosages of the compounds of the present invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known in the art; for example,see U.S. Pat. No. 4,938,949.

Modes of administration of the compositions used in the invention areexemplified below. However, the compositions can be delivered by any ofa variety of routes including: by injection (e.g., subcutaneous,intramuscular, intravenous, intra-arterial, intraperitoneal), bycontinuous intravenous infusion, cutaneously, dermally, transdermally,orally (e.g., tablet, pill, liquid medicine, edible film strip), byimplanted osmotic pumps, by suppository or aerosol spray. Routes ofadministration include, but are not limited to, topical, intradermal,intrathecal, intralesional, intratumoral, intrabladder, intravaginal,intra-ocular, intrarectal, intrapulmonary, intraspinal, dermal,subdermal, intra-articular, placement within cavities of the body, nasalinhalation, pulmonary inhalation, impression into skin andelectroporation.

Depending on the route of administration, the volume of a compositioncomprising MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC in an acceptablecarrier, per dose, is about 0.001 ml to about 100 ml. In one embodiment,the volume of a composition comprising MRNC, MpRNC, MbRNC, MsRNC, MapRNCand MvRNC in an acceptable carrier, per dose is about 0.01 ml to about50 ml. In another embodiment, the volume of a composition comprisingMRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC in an acceptable carrier,per dose, is about 0.1 ml to about 30 ml. A composition comprising MRNC,MpRNC, MbRNC, MsRNC, MapRNC and MvRNC in an acceptable carrier may beadministered in a single dose treatment or in multiple dose treatments,on a schedule, or over a period of time appropriate to the disease beingtreated, the condition of the recipient and the route of administration.The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

Methods for Activating Immune System Receptors

The present invention provides methods for activating immune systemreceptors comprising administering to a subject in need thereof atherapeutically effective amount of MRNC, MpRNC, MbRNC, MsRNC, MapRNCand MvRNC and a pharmaceutically acceptable carrier. Immune systemreceptors that can be activated by the present invention are (but notlimited to) Nucleotide-binding oligomerization domain 2 (NOD2), appattern recognition receptor (PRR) responsible for sensing thepathogen-associated molecular pattern (PAMP) molecule muramyl dipeptide,the minimal structural component of bacterial peptidoglycan with immunestimulant activity, and Toll-like receptor 2 (TLR2), a ppatternrecognition receptor (PRR) responsible for sensing a wide range ofpathogen-associated molecular pattern (PAMP) molecules such aspeptidoglycan, lipomannan (LM), lipoarabinomannan (LAM), andlipoproteins and lipopeptides containing a (palmitic acid)_(n)-derivedN-terminus cysteine or cysteines. The activation of these two immunesystem receptors is known by those of ordinary skill in the art toresult in the generation of anticancer activity and anti-infectiveactivity (including but not limited to bacterial, fungal and viralinfections), as well as a stimulation of vaccine adjuvant activity andthe differentiation of human bone marrow CD14⁺ cells. The ability ofMRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC to function as an agonistfor both of these receptors provides an unmet need and further providessignificant advantages over monofunctional NOD2 or TLR2 agonists thatare used as therapeutic agents in the fields of oncology, infectiousdiseases or vaccines.

Method for Treating Diseases Including Cancer and Disorders of theImmune System

The present invention provides methods for treating cancer comprisingadministering to a subject in need thereof a therapeutically effectiveamount of MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC and apharmaceutically acceptable carrier.

In another aspect, the methods and compositions of the invention areuseful in the therapeutic treatment of cancer, and diseases or disordersof the immune system. In yet a further embodiment, some cancers can beprevented by the timely administration of the compositions as aprophylactic, prior to onset of symptoms, or signs, or prior to onset ofsevere symptoms or signs of a cancer disease. Thus, a patient at riskfor a particular cancer disease can be treated with one or more of thecomposition of the present invention as a precautionary measure.

In yet another aspect, the present invention is directed to a method ofrelieving or ameliorating cancer or tumor development, metastasis,cellular proliferation in cancer cells, and/or inhibition of symptomsassociated with any one or more of the above-identified cancer diseasesand/or cancer indications in a subject suffering from any one or more ofthe above-identified cancer diseases or cancer indication. This methodcomprises administering to the subject in need thereof of atherapeutically effective amount of MRNC, MpRNC, MbRNC, MsRNC, MapRNCand MvRNC in a pharmaceutically acceptable carrier. The MRNC, MpRNC,MbRNC, MsRNC, MapRNC and MvRNC may be administered with apharmaceutically acceptable carrier, either alone or in combination withone or more anti-inflammatory compounds, immune stimulatory agents, oranti-cancer agents, wherein the MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC is sufficient to inhibit the cancer or tumor development,metastasis, and/or cell proliferation in cancer cells.

MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are effective as therapeuticagents for treating, abating or eliminating a disease including, but notlimited, to a cancer. Cancers include, but are not limited to, primaryor metastatic cancers, squamous cell carcinoma, fibrosarcoma, sarcoidcarcinoma, melanoma, mammary cancer, lung cancer, colorectal cancer,renal cancer, osteosarcoma, cutaneous melanoma, basal cell carcinoma,pancreatic cancer, bladder cancer, cancer of the urethra, cervicalcancer, endometrial cancer, ovarian cancer, prostate cancer, leukemia,lymphoma and metastases derived there from.

In one aspect, the cancer diseases and cancer disorders that may betreated using the MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCcompositions of the present invention include for example, but notlimited to, AIDS-associated cancers, bladder cancer, bone cancer, brainand spinal cord cancers, metastatic brain tumors, pediatric braintumors, breast cancer, male breast cancer, cervical cancer, colorectalcancer, endometrial and other uterine cancers, esophageal cancer,gallbladder and bile duct cancers, gastric (stomach) cancer, head andneck cancers, kidney cancer, leukemia, liver cancer, liver metastases,lung cancer, lymphomas, mammary adenocarcinoma, melanoma, multiplemyeloma, myelodysplastic syndrome, neuroblastoma, osteosarcoma, ovariancancer, pancreatic cancer, pediatric cancers, pituitary tumors, prostatecancer, hematological disorders, sarcoma, solid tumors, retinoblastoma,skin cancer, soft-tissue sarcoma, testicular cancer, thyroid cancer,uterine cancers, Wilms' tumor, bronchus cancer, colon and rectum cancer,urinary cancer, non-Hodgkin lymphoma, melanomas of the skin, kidney andrenal cancer, pelvis cancer, pancreatic cancer, oral cavity cancer andpharynx cancer, stomach esophagus cancer, intrahepatic bile duct cancer,larynx cancer, acute myeloid leukemia, chronic lymphocytic leukemia,soft tissue cancer including heart, GIST (gastro-intestinal stromaltumors), small intestine cancer, chronic myeloid leukemia, acutelymphocytic leukemia, cancer of the anus and anal canal, anorectalcancer, vulva cancer, pleura cancer, malignant mesothelioma, cancer ofthe bones and joints, hypopharynx cancer, eye and orbit cancer, nose andnasal cavity cancer, middle ear cancer, nasopharynx cancer, ureteralcancer, peritoneum cancer, omentum and mesentery cancer, andgastrointestinal carcinoid tumors or any combination thereof

In yet another aspect, the cancer diseases and cancer disorders that maybe treated using the present invention include, but are not limited to,colorectal cancer, gastric cancer, ovarian cancer, osteosarcoma,hepatocellular carcinoma, Burkitt's lymphoma, primary effusionlymphomas, angioimmunoblastic lymphadenopathy, acquired immunedeficiency syndrome (AIDS)-related lymphoma, T-cell lymphomas, oralhairy leukoplakia, lymphoproliferative disease, lymphoepithelialcarcinoma, body-cavity-based lymphoma or B-cell lymphomas,non-keratinizing carcinoma, squamous cell nasopharyngeal carcinoma,kidney transplant-associated epithelial tumors, angiosarcoma, Kaposi'ssarcoma, angiolymphoid hyperplasia, prostatic neoplasm, retinoblastoma,Li-Fraumeni syndrome, Gardner's syndrome, Werner's syndrome, nervoidbasal cell carcinoma syndrome, neurofibromatosis type 1, cervicaldysplasia, neuroblastoma, primary macroglobulinemia, insulinoma, mycosisfungoides, osteogenic sarcoma, premalignant skin lesions (topical),rhabdomyosarcoma, osteogenic, polycythemia vera, essentialthrombocytosis or any combination thereof. MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC, may be used as anti-infective agents. The invention isalso useful for protecting animals upon exposure to pathogenicmaterials, such as viruses and bacteria. The present invention is usefulfor regulating mammalian hematopoiesis or inducing hematopoietic andmyeloid recovery (leucopenia, neutropenia, thrombocytopenia or anemia)in animals with cancer recovering from irradiation, surgery orchemotherapeutic treatment. The present invention is useful for theprevention or treatment of diverse hematopoietic and myeloidabnormalities associated with medications or diseases, such as, but notlimited to the acquired immunodeficiency syndrome (AIDS),myelodysplastic syndromes, autoimmune diseases, end-stage renal diseasesor viral infections.

The present invention is also useful for the treatment or abatement ofautoimmune disorders, inflammatory and infectious diseases. MRNC, MpRNC,MbRNC, MsRNC, MapRNC and MvRNC can be used to treat a wide variety ofinfections caused by viruses, bacteria, mycobacteria (such as but notlimited to Mycobacterium tuberculosis or Mycobacterium avium subspeciesparatuberculosis), or intracellular organisms including, but not limitedto, infection by herpes virus such as equine rhinopneumonitis,infectious bovine rhinotracheitis, endometritis, herpes simplex, herpeszoster, ocular herpes, feline viral rhinotracheitis, and a herpes viruswhich infects the respiratory tracts of cats. The invention also iseffective in the treatment of parvovirus infections of young dogs. Thecompositions of the present invention are efficacious as a therapeuticagent for genital herpes infections and acquired immune deficiencysyndrome of humans, as well as other viral infections of animals andhumans. For example, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC can beused to treat viral, bacterial, protozoa and fungal infections, such as,but not limited to, Equine Herpes Virus, Equine Influenza Virus, Herpessimplex, Streptococcal species such as but not limited to Streptococcuszooepidemicus, E. coli, and Llama Babesia

The following examples will serve to further illustrate the presentinvention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims. It is to be understood that one of skill in the art, will uponreading the detailed description and the examples of the presentinvention apply the findings therein to the preparation of immunestimulatory and anti-cancer RNC from Gram-positive and Gram-negativebacteria without departing from the spirit and intent of the presentinvention or the scope of the appended claims.

Example 1 Cultivation of Mycobacteria in Synthetic Medium

The preparation of mycobacterial cell wall compositions as taught inU.S. Pat. No. 4,744,984 and U.S. Pat. No. 6,326,357 specified the use ofBACTO™ AC broth (Difco Labs, now Becton Dickenson) for the preparationof mycobacterial cell mass. This broth contains proteose peptone No. 3,beef extract, yeast extract and malt extract (Difco™ & BBL™ Manual ofMicrobiological Culture Media, second edition, 2009, pp 35-36). Inaddition, seed stocks of Mycobacterium phlei used as the example inthese patents were stored in bovine milk prior to generation of biomass.The aforementioned patents also teach that cultivation is static, andthat the mycobacterial cultures grow as a surface pellicle on theculture medium.

In order to eliminate the potential for contamination of mycobacterialbiomass by the aforementioned substances, a novel synthetic medium wasdeveloped for the cultivation of mycobacteria. Middlebrook 7H9 broth isknown to those of ordinary skill in the art to be suitable for thecultivation of pure cultures of mycobacteria but requires the additionof supplements containing animal sourced material (Middlebrook ADCenrichment containing bovine serum albumin and catalase) andadditionally benefits from the use of polysorbate 80 or glycerol (Difco™& BBL™ Manual of Microbiological Culture Media, second edition, 2009, pp355-356). However, it was unexpectedly found that efficient growth ofmycobacteria occurred in Middlebrook 7H9 without the use of theexogenous proteins bovine albumin or catalase that are found inMiddlebrook ADC enrichment medium, or of the non-ionic detergentpolysorbate 80. It was also unexpectedly found that the use ofadditional iron (as ferric ammonium citrate) and a source of additionalcarbon and nitrogen (such as but not limited to asparagine or ammoniumcitrate) resulted in superior yields of mycobacterial mass when comparedto cultivation with Middlebrook 7H9 broth alone. The use of additionalcarbon, nitrogen and iron in an appropriate ratio is used to facilitatemetabolic activity and cell division through optimization of the carbonto nitrogen ratio for the synthesis of proteins, nucleic acids, andother cellular constituents, as well as enhancing metabolism throughincreased iron availability, thus increasing the mycobacterial divisionrate and mycobacterial cell mass.

The following results serve to demonstrate the impact of changing theC:N:Fe ratio in the cultivation medium on the growth of Mycobacteriumphlei, used herein as an example of a representative mycobacterialspecies. It is to be realized that the findings described below may bereplicated by using alternate sources of C, N and Fe, and can be appliedto other mycobacteria without departing from the scope and intent of thepresent invention.

The composition of Middlebrook 7H9 broth and ADC is available in forexample the Difco and BBL Manual of Microbiological Culture Media,2^(nd) Edition, 2009, pages 355-356. The growth of Mycobacterium phleiwas determined in the following newly developed cultivation media, whichare defined hereafter as Mycobacterial Culture Media Compositions(MCMC):

-   A. MCMC composition containing Middlebrook 7H9 (BD Canada,    Mississauga, Ontario), glucose and glycerol.-   B. MCMC composition containing Middlebrook 7H9 glucose, glycerol and    additional Fe (as ferric ammonium citrate) and asparagine as iron,    carbon sources and nitrogen sources.-   C. MCMC composition containing glucose, glycerol and containing Fe    (as ferric ammonium citrate), asparagine and ammonium citrate    (dibasic) as iron, carbon and nitrogen sources.-   D. MCMC composition containing glucose, glycerol, Fe (as ferric    ammonium citrate) and ammonium citrate (dibasic) as iron, carbon and    nitrogen sources.

In a separate study the impact of an MCMC composition containing ironand asparagine as the L, DL or D stereoisomer was also determined usingComposition C described above.

To prepare MCMC A and MCMC B, Middlebrook 7H9 broth, 9.4 g ofMiddlebrook 7H9 powdered media was combined with the additives with theexception of glucose, combined with glycerol and additives, dissolved inwater (900 mL) and sterilized by steam autoclaving. Glucose wasdissolved in water and always filter-sterilized separately and addedafterwards to give a final volume of 1000 mL. MCMC C and MCMC D do notcontain Middlebrook 7H9 powdered medium. Table 1 shows the mol contentof C, N and Fe, per liter cultivation media for Middlebrook 7H9 brothcontaining glycerol, glucose and the additives described above, as wellas the two cultivation media that do not contain Middlebrook 7H9 brothpowder.

TABLE 1 C, N and Fe content of synthetic media used to preparemycobacterial cell mass MCMC Carbon* Nitrogen** Iron*** compositionmMol/L Ratio mMol/L C:N Ratio mM/L C:Fe Ratio MCMC-A 769.30 1 7.33  9.5× 10⁻³ 0.14 1.8 × 10⁻⁴ MCMC-B 792.65 1 18.80 23.71 × 10⁻³  0.25 3.2 ×10⁻⁴ MCMC-C 938.05 1 61.22 61.2 × 10⁻³ 0.25 2.7 × 10⁻⁴ MCMC-D 915.33 149.86 54.5 × 10⁻³ 0.25 2.7 × 10⁻⁴ MCMC-C (D or L asparagine) 938.05 161.22 61.2 × 10⁻³ 0.25 2.7 × 10⁻⁴ *All available (metabolically) carbonhas been used in the calculation. **All available (metabolically)nitrogen has been used in the calculation. ***All available iron hasbeen used in the calculation. Ferric ammonium citrate is defined asC₆H₁₀FeNO₈

Middlebrook 7H9 powder was obtained from BD Canada, Mississauga, Ontarioand is known to one of ordinary skill in the art. The approximateformula for making about 900 mL of Difco™ Middlebrook 7H9 Broth is asfollows: Ammonium Sulfate (0.5 g), L-Glutamic Acid (0.5 g), SodiumCitrate (0.1 g), Pyridoxine (1.0 mg), Biotin (0.5 mg), DisodiumPhosphate (2.5 g), Monopotassium Phosphate (1.0 g), Ferric AmmoniumCitrate (0.04 g), Magnesium Sulfate (0.05 g), Calcium Chloride (0.5 mg),Zinc Sulfate (1.0 mg), Copper Sulfate (1.0 mg).

All other reagents were from Sigma, Oakville, Ontario. The compositionin g/L of the cultivation media used to prepare MCMC-A, MCMC-B, MCMC-Cand MCMC-D are shown below. However it is to be understood that whileeach specific number listed is for one recipe of MCMC-A, MCMC-B, MCMC-Cand MCMC-D, each of these numbers may be increased or decreased by about10% to about 20% to make variations of the MCMC-A, MCMC-B, MCMC-C andMCMC-D cultivation media, with corresponding changes in the C:N and C:Feratios.

MCMC-A. Middlebrook 7H9 powder 9.5 g Glycerol 2 mL Glucose 20 gDistilled water to 1 L MCMC-B Middlebrook 7H9 powder 9.5 g DL-Asparagine0.75 g Ferric ammonium citrate 0.03 g Glycerol 2 mL Glucose 20 gDistilled water to 1 L MCMC-C Ammonium citrate 4.84 g DL-Asparagine 0.75g L-Glutamic acid 1.0 g Pyridoxine 0.002 g Biotin 0.0005 g Disodiumphosphate 5 g Monopotassium phosphate 2.0 g Ferric ammonium citrate 0.12g Magnesium sulfate 0.25 g Calcium chloride 0.0005 g Zinc sulfate 0.005g Oleic acid 0.125 g Glycerol 2 mL Glucose 20 g Distilled water to 1 LMCMC-C (D-asparagine) Ammonium citrate 4.84 g D-Asparagine 0.75 gL-glutamic acid 1.0 g Pyridoxine 0.002 g Biotin 0.0005 g Disodiumphosphate 5 g Monopotassium phosphate 2.0 g Ferric ammonium citrate 0.12g Magnesium sulfate 0.25 g Calcium chloride 0.0005 g Zinc sulfate 0.005g Oleic acid 0.125 g Glycerol 2 mL Glucose 20 g Distilled water to 1 LMCMC-C (L asparagine) Ammonium citrate 4.84 g L-Asparagine 0.75 gL-glutamic acid 1.0 g Pyridoxine 0.002 g Biotin 0.0005 g Disodiumphosphate 5 g Monopotassium phosphate 2.0 g Ferric ammonium citrate 0.12g Magnesium sulfate 0.25 g Calcium chloride 0.0005 g Zinc sulfate 0.005g Oleic acid 0.125 g Glycerol 2 mL Glucose 20 g Distilled water to 1 LMCMC-D Ammonium citrate 4.84 g L-glutamic acid 1.0 g Pyridoxine 0.002 gBiotin 0.0005 g Disodium phosphate 5 g Monopotassium phosphate 2.0 gFerric ammonium citrate 1.2 g Magnesium sulfate 0.25 g Calcium chloride0.0005 g Zinc sulfate 0.005 g Oleic acid 0.125 g Glycerol 2 mL Glucose20 g Distilled water to 1 L

Glucose 20 g Distilled water to 1 L MCMC-D Ammonium citrate 4.84 gL-glutamic acid 1.0 g Pyridoxine 0.002 g Biotin 0.0005 g Disodiumphosphate 5 g Monopotassium phosphate 2.0 g Ferric ammonium citrate 1.2g Magnesium sulfate 0.25 g Calcium chloride 0.0005 g Zinc sulfate 0.005g Oleic acid 0.125 g Glycerol 2 mL Glucose 20 g Distilled water to 1 L

The following procedure describes the preparation of mycobacterial cellmass from Mycobacterium phlei, and therefore serves as a general examplefor the preparation of mycobacterial cell mass from mycobacteria ingeneral. It is recognized that one of ordinary skill in the art onreading this example will appropriately modify the procedures to addressthe specific needs of individual mycobacterial species, complexes andstrains.

Mycobacterium phlei (strain 110) was stored as colonies on Petriaganislants at 4° C. or as a suspension in Middlebrook 7H9 containing 20%glycerol at −80° C. Determination of optimal cultivation carbon,nitrogen and iron ratios was conducted using seed cultures prepared fromPetriagani slants. Colonies from the Petriagani slant were placed in 1.2L of the M Medium and after dispersion were cultivated for 7 days (250rpm, 37° C. in an orbital shaker, Lab-Line Instruments, Melrose Park,Ill., USA). After adjustment to a standardized OD, 200 mL were added toa series of culture flasks containing 1 L of the respective MCMC.Cultivation was continued (250 rpm, 37° C. in an LAB-Line orbitalshaker), and replicate flasks were removed at intervals over a period of10 days. Mycobacterial mass was isolated by low speed centrifugationwashing, lyophilized and the dry weight determined. Where lyophilizationwas impractical, dry weight was calculated from wet cell mass using theconversion factor wet cell mass/6.81=dry cell mass (the mean±SD for thisfactor was previously determined by experimentation to be 6.81±1.16,n=7).

The results in Table 2 show the impact of different carbon, nitrogen andiron ratios in the MCMC on the yield of Mycobacterium phlei, as well asthe time of optimal yield in days, and the results in Table 3 shows theimpact of using racemic DL-, D- or L-asparagine on the yield ofmycobacterial cell mass. It is to be understood that these studies wereconducted at different times, and that the absolute yields ofmycobacterial cell mass vary from study to study.

TABLE 2 Impact of C, N and Fe content on yield of Mycobacterium phleicell mass MCMC Yield, g/L* (fold increase Composition versus A) Day A4.52 10 B 4.16 (0.9) 6 C 6.24 (1.4) 6 D 6.20 (1.4) 4 *Yield is expressedas g dry weight mycobacterium phlei per liter of culture broth.

TABLE 3 Impact of asparagine stereoisomer D, L or a DL racemic mixtureon the yield of Mycobacterium phlei MCMC Yield, g/L (fold increaseComposition versus DL asparagine) Optimal yield, day C usingDL-asparagine 4.9 6 C using D-asparagine 4.9 (0)  6 C using L-asparagine5.73 (1.2) 6

The results shown in Table 2 demonstrate that increasing the amount ofavailable carbon, nitrogen and iron gives rise to increases in the yieldof cell mass, as well as decreasing the time required to obtain maximalyield. The results also show that the use of, for example, but notlimited to asparagine or ammonium citrate (i.e., an available source ofnitrogen, whether it is in the form of an organic or inorganic molecule)increases the yield. Two advantages associated with the use of ammoniumcitrate rather than ammonium sulfate, are the relatively slow metabolismof the carbons of citrate (the salt of a weak acid, which providessubstantial buffering capacity to the medium) and the observed reducedfluctuations in the pH of the broth during cultivation that areconsistent with the generation of sulfuric acid from thenon-metabolizable sulfate (the salt of a strong acid).

The results shown in Table 3 demonstrate that the use of D-asparagine ora racemic mixture if DL asparagine gave lower mycobacterial cell massyield than when the L-stereoisomer was used. These data are consistentwith a more efficient utilization of the L-stereoisomer of asparaginerather than the D-stereoisomer or the racemic mixture. Ammonium citratewould appear to possess a further advantage as a source of nitrogen inthat the ammonium ions are not racemic.

A third study was conducted using the optimized MCMC-C whereMycobacterium phlei and Mycobacterium smegmatis yields were compared.Mycobacterium smegmatis (ATCC, Manassas, Va., strain ATCC14468) wasprepared as described for Mycobacterium phlei, and head-to-headcultivations were performed as described above.

TABLE 4 Use of MCMC-C for the preparation of Mycobacterium phlei andMycobacterium smegmatis cell mass Mycobacterium Yield, g dry weight/LOptimal yield, day Mycobacterium phlei 7.6 6 Mycobacterium smegmatis 9.66

The results shown in Table 4 demonstrate that the use of the MCMCcomposition C results in significant quantities of mycobacterial cellmass in a relatively short period of time, and that such yields are notrestricted to Mycobacterium phlei

A separate study was performed in order to determine the effect ofadditional aeration of the medium during cultivation. Cultivation ofMycobacterium phlei was carried out as described above, except thattripled baffled Fermbach flasks were used in order to increase mixing ofthe medium and access to atmospheric oxygen. The growth of Mycobacteriumphlei in MCMC-C. medium was compared with its growth in Bacto™ AC brothand MCMC-A. Mycobacterium phlei cell mass yield at 120 hours wasdetermined as g dry weight mycobacterial cell mass/liter cultivationmedium. The results showed that using Bacto™ AC broth the yield was 5.0g/L, using MCMC-A the yield was 3.15 g/L, and using MCMC-C the yield was9.2 g/L (2.9-fold increase versus MCMC-A and 1.84-fold increase versusBacto™ AC broth). These data demonstrate that under cultivationconditions that increase mixing and aeration (such as but not limited toFermbach flasks) there are further increases in the yield ofMycobacterium phlei, and that the yield in the new medium composition isgreater than that found with either Bacto™ AC broth or MCMC-A.

Preparation of mycobacterial cell wall RNC of the present invention wasconducted using seed cultures of mycobacteria prepared from frozenstocks. A seed culture was prepared from frozen stock by adding 1 vialof the frozen stock to 600 mL of the modified culture medium, andcultivation was carried out for 7 days at 37° C. with agitation in anorbital shaker at 160-250 rpm (Lab-Line, Melrose Park, Ill., USA). Theseed culture was then used to inoculate a larger culture volume at a1:10 dilution, and cultivation carried out using the same conditions for7 days. Mycobacterial biomass was harvested by low speed centrifugalwashing, after which the pelleted mycobacteria were resuspended in waterfor injection (nuclease-free) to give a 10% w/v suspension. Thecompositions of the present invention were prepared from this suspensionas described in the Examples below.

Typically, the cultivation of Mycobacterium phlei as described in U.S.Pat. No. 6,326,357 takes between 7-10 days for the generation of a seedculture and an additional 10 to 20 days for the generation ofmycobacterial cell biomass (cultivation total time between 17-30 days).The yield of this manufacturing process is approximately 20 g wet weightMycobacterium phlei/liter of culture medium, corresponding to a dryweight of approximately 2-3 g Mycobacterium phlei/liter. The newmanufacturing process described above using the MCMC compositions andcultivation conditions takes 14 days in total, and the yield isapproximately 6.5-7.6 g dry weight Mycobacterium phlei/liter, thusproviding a significant decrease in the time required to prepare thebiomass in addition to a significant increase in the yield ofmycobacterial cell mass. Such improvements provide unexpected increasesin biomass production efficiency when compared to previous teachings.

It is to be realized that the important principles described in thisexample are the use of a cultivation medium for the preparation ofmycobacterial cell mass that does not contain exogenous animal, plant orfungal material, and a cultivation process that optimizes the growth ofthe mycobacteria through the use of an optimized medium in conjunctionwith agitation to maintain the mycobacteria in suspension, rather thanas a pellicle that is characteristic of stationary growth cultivationconditions. Modification to these principles may be made by one ofordinary skill in the art after reading this example that result inoptimal growth of a particular mycobacterial species, complex or strainwithout departing from the spirit of the present invention and/or thescope of the appended claims.

Example 2 Controlling the Number of Intact Mycobacteria During thePreparation of Immune Stimulatory and Anticancer Compositions

The following example identifies new procedures for the preparation ofthe compositions of the present invention that use working volumes thatare scalable, range from several mL to multi-liter volumes, and thatresult in the efficient production of new bacterial compositionscomprising nucleic acids and cell walls.

Mycobacterial cell mass (Mycobacterium phlei is used as an illustrativeand representative example of mycobacterial cell mass) was prepared asdescribed in Example 1. After pelleting by low-speed centrifugation toremove culture medium components, M. phlei cells (prepared as a 10% w/vsuspension in DNase- and RNase-free water for injection) was disruptedby high-pressure homogenization (18,000 pounds per square inch [PSI], 5cycles, at a temperature of 4° C.) using an Avestin EmulsiFlex-05high-pressure homogenizer (Avestin, Ottawa, Ontario, Canada). Resort maybe made to similar or equivalent systems without departure from thepresent invention. The homogenate was then centrifuged at a relativecentrifugal force identified as being optimal for the removal of intactmycobacteria (3,160×g for 30 min at 4° C.). In this example, samples ofthe 10% w/v M. phlei suspension, the homogenate after high-pressurehomogenization, the supernatant from the low-speed centrifugation(containing intact and contaminating M. phlei) and the supernatant fromthe low-speed centrifugation that had been subjected to additionalcycles of high-pressure homogenization were serially diluted (dilutionsof 10⁻² to 10⁻⁷ in water for injection) and the number of colony-formingunits (CFU) determined by plating on tryptic soy agar growth media,incubating for 72-96 h at 37° C., and counting the colonies andexpressing them as CFU/mL.

The Avestin high-pressure homogenization system has been reported toachieve ˜100% disruption of yeast and Escherichia coli cells after 1-3cycles. The failure of 5 homogenization steps to achieve 100% disruptionof Mycobacterium phlei would indicate to those of skill in the art thatthis would be the limit of efficiency for this microorganism.Unexpectedly however it was found that subjecting the low-speedcentrifugation supernatant containing intact Mycobacterium phlei cellsthat were apparently resistant to 5 cycles of high-pressurehomogenization to additional steps (2-3) of high-pressure homogenizationat 18,000 PSI at a temperature of 4° C. resulted in further reductionsin intact Mycobacterium phlei cells (Table 5). The unexpected benefitsof additional high-pressure homogenization steps are further controlledreductions in the number of intact Mycobacterium phlei cells and anincrease in the yield of mycobacterial cell wall-RNC.

As Mycobacterium phlei grows in aggregates of cells in suspensionculture, it is impossible to directly determine the number of CFUs inthe mycobacterial suspension (determined to be an approximately 100-foldunderestimation). Use was made of a calculated estimation determined asfollows: The size of the microorganism as shown in FIG. 1 is similar tothat of Escherichia coli (0.5×2 μm: Imaging whole Escherichia colibacteria by using single-particle x-ray diffraction. Miao J, et al. PNAS2003, 100:110-112), thus allowing a calculation of the estimated CFU ofa 10% w/v suspension of Mycobacterium phlei based on the weight ofindividual Escherichia coli, which is 665 femtograms (fg) (Single celldetection with micromechanical oscillators. Illic, B, et al., J. Vac.Sci. Technol. B. 2001, 19(6):2825-2828)

TABLE 5 Effect of high pressure homogenization on the number of intactmycobacteria Sample CFU/mL 10% w/.v suspension (calculated 1.5 × 10¹¹estimation) High-pressure homogenization (5 cycles) 8.7 × 10⁸ Low-speedcentrifugation supernatant 3.0 × 10⁷ Low-speed centrifugationsupernatant 1.9 × 10⁷ following by 3 additional high-pressurehomogenization cycles

The results shown in Table 5 demonstrate that the use of 5 cycles ofhigh-pressure homogenization is consistent with the efficient disruptionof the mycobacteria in the suspension (99.42%—based on the abovecalculation). The low-speed centrifugation after 5 cycles ofhigh-pressure homogenization resulted in a 5,000-fold reduction in thenumber of intact, viable mycobacterial cells as compared to anequivalent M. phlei sample that had not undergone high-pressurehomogenization. The use of further high-pressure homogenization stepsusing the supernatant from the low-speed centrifugation resulted in anadditional 0.007% content representing a 2,895-fold reduction in thenumber of intact, viable mycobacterial cells as compared to anequivalent Mycobacterium phlei samples that had not undergonehigh-pressure homogenization.

It is to be realized that the important principles demonstrated in thisexample are the unexpected benefits arising from the use of additionalhomogenization steps are further disruption of intact mycobacteria,which as Gram-positive microorganisms are considered to be resistant tohigh-pressure homogenization, and the ability to control through the useof differing homogenization steps the level of intact mycobacteria inthe mycobacterial cell wall RNC fraction contained in the low-speedcentrifugation supernatant.

Example 3 Preparation of Compositions Comprising Mycobacterial CellWalls and RNA (MpRNC)

To further demonstrate the utility of the new approach described aboveto prepare mycobacterial cell wall compositions, new procedures weredeveloped that required the use of different homogenization pressuresand number of homogenization cycles that enabled the preparation of newmycobacterial cell wall-RNA compositions (MRNC) formulated withmycobacterial cell walls and comprising: a) a controlled reduction inthe intact mycobacterial cell content; b) the generation ofoligoribonucleotides and polyribonucleotides of defined length, and c)the generation of unexpected immune stimulant activity.

In this example mycobacterial cell mass (Mycobacterium phlei is used asan illustrative and representative example of mycobacterial cell mass,and it is to be realized that the manufacturing procedures described inthis example are applicable to both bacteria and mycobacteria) wasprepared as described in Example 1. Intact Mycobacterium phlei cellswere first washed by low-speed centrifugation to remove culture mediumcomponents, and were then disrupted using high-pressure homogenizationwith an Avestin EmulsiFlex-C5 high-pressure homogenizer (Avestin,Ottawa, Ontario, Canada). After high-pressure homogenization theremaining intact mycobacterial cells were removed by differentialcentrifugation using relative centrifugal forces that were optimized forthe controlled removal of any residual, intact and undisruptedmycobacteria as well as the elimination of soluble material (cytoplasmiccontents including proteins). The intact mycobacterial cell-freepreparation, comprising the RNA of the mycobacterium in the form ofoligoribonucleotides and polyribonucleotides associated withmycobacterial cell wall fragments, was further purified bycentrifugation washing at a higher relative centrifugal force, in orderto remove soluble contaminants. The MpRNC fraction was isolated as apellet following centrifugation washing at high relative centrifugalforce. The number of live mycobacteria in the MpRNC fraction wasdetermined by plating serial dilutions of the mycobacterial intactcell-free fraction on tryptic soy agar growth media, incubating for72-96 h at 37° C., and determining the number of colony forming units(CFUs). The MpRNC suspension was then heat-treated at 121° C. forbetween 5-30 min. Table 6 shows the steps used to prepare MpRNC with ahigh-, an intermediate-, or with a low intact mycobacterial cell content(hereafter referred to as MpRNC High, MpRNC Intermediate and MpRNC Low,respectively).

TABLE 6 Preparation of MpRNC containing High, Intermediate and Lowlevels of intact Mycobacterium phlei Step MpRNC High MpRNC IntermediateMpRNC Low 1 High-pressure High-pressure High-pressure homogenization,homogenization, homogenization, 10,000 PSI, 18,000 PSI, 27,000 PSI, 2cycles 5 cycles 5 cycles 2 High-speed Low-speed Low-speedcentrifugation, 38,400 x centrifugation, 3,160 x centrifugation, 3,160 xRCF, to pellet MpRNC RCF to remove intact RCF to remove intact and anyremaining intact cells cells cells 3 High-pressure High-speedHigh-pressure homogenization, 10,000 centrifugation, 38,400 xhomogenization, 27,000 psi, of resuspended RCF to pellet MpRNC psi, tofurther reduce MpRNC pellet and any remaining intact intact cell content2 cycles cells 3 cycles 4 MpRNC High High-pressure High-speedhomogenization, 18,000 centrifugation, 38,400 x psi, of resuspended RCFto pellet MpRNC MpRNC pellet and any remaining intact 2 cycles cells 5MpRNC Intermediate High-pressure homogenization, 27,000 psi, ofresuspended MpRNC pellet 2 cycles 6 MpRNC Low

The results shown in Table 7 show the number of CFUs in MpRNC preparedusing the various procedures described in Table 6.

TABLE 7 Preparation of MpRNC with differing levels of intactmycobacterial cell content Differential Mycobacterial % IntactHomogenization centrifugation CFU/mg mycobacteria MpRNC pressure, psiRCF (low; high) MpRNC (w/w)* A) High 10,000, 4 cycles  None; 38,400 2.94× 10⁸ 19.6% Mycobacterium phlei (2 before high-speed cell contentcentrifugation, 2 after) B) Intermediate 18,000, 7 cycles 3,160; 38,4001.00 × 10⁷ 0.66% Mycobacterium phlei (5 before low-speed cell contentcentrifugation, 2 cycles after high- speed centrifugation) C) Low27,000, 10 cycles 3,160; 38,400 2.88 × 10⁶ 0.19% Mycobacterium phlei (5before low-speed cell content centrifugation, 3 cycles after low- speedcentrifugation, 2 cycles after high- speed centrifugation) *Based on anintact bacterial weight of 665 fg/bacterium (Single cell detection withmicromechanical oscillators. Illic, B, et al., J. Vac. Sci. Technol.2001, B. 19(6): 2825-2828)

The data show that by increasing the homogenization pressure and thenumber of homogenization cycles that the MpRNC undergoes a significantand more importantly a controlled reduction in the number of viableintact Mycobacterium phlei cells present in the MpRNC. Calculation ofthe % weight of intact mycobacteria (based on a bacterial weight of 665fg) showed a controlled range of <0.2% (MpRNC low) to 19.6% (MpRNChigh). Those of skill in the art will recognize that the number ofviable mycobacterial cells in the MpRNC can therefore be readilycontrolled over a 100-fold range by the appropriate use of a definedmethod, such as the controlled use of homogenization pressure, thenumber of homogenization cycles and the use of differentialcentrifugation as described in the present invention.

Example 4 Preparation of MRNC from Mycobacterium bovis Strain BCG,Mycobacterium smegmatis and Mycobacterium Vaccae

In this example, three different mycobacteria, one known to be slowgrowing in culture and two known to be fast growing in culture, wereused to prepare different MRNC compositions. As such, this example inconjunction with Examples 1, 2 and 3 of the present invention teachesone of skill in the art how to prepare MRNC from any mycobacterialspecies or strain. In order to preserve the nucleic acid compositionnuclease-free (RNase-free and DNase-free) reagents were used throughout.One of ordinary skill in the art on reading the present invention willalso recognize that application of the use of different homogenizationcycles and pressures, and the selective use of differentialcentrifugation, as described in detail in Examples 1, 2 and 3 for thepreparation of MpRNC from Mycobacterium phlei, enables the preparationof RNC compositions containing the optimal level of intact mycobacterialcells from other species for the desired application.

Mycobacterium bovis BCG from mycobacterial biomass or from commerciallyavailable sources can be used to prepare MbRNC compositions. In thisexample the Connaught strain of BCG was used. BCG is availablecommercially as a lyophilized power containing 2-10×10⁸ CFU/mg(Immucyst, Aventis, Toronto, ON, Canada). The lyophilized powder wasfirst resuspended in water for injection (Wisent) for 30 min 20° C. withintermittent vortexing. The suspended cells were pelleted by low-speedcentrifugation and then disrupted using high-pressure homogenizationwith an Avestin EmulsiFlex-05 high-pressure homogenizer followed bydifferential centrifugation to isolate MbRNC as described for MpRNCIntermediate with minor modification. Specifically, a 0.243% w/vMycobacterium bovis BCG suspension in water for injection was subjectedto 5 cycles of high-pressure homogenization at 18,000 psi with a heatexchanger circulating water at 4° C. The homogenate was then centrifugedat 3,160×g for 30 min to sediment any residual, intact and undisruptedMycobacterium bovis BCG. The supernatant, containing RNC of themycobacterium associated with mycobacterial cell wall fragments, wasthen centrifuged at 38,400×g for 1 hour to remove soluble contaminantsand to pellet RNC and Mycobacterium bovis BCG cell walls (MbRNC). Thepellet was then re-suspended in water for injection and was homogenizedfor two additional cycles as described above. The suspension containingMycobacterium bovis MbRNC was heat-treated at 121° C. for between 5-30min.

Mycobacterium smegmatis (obtained from the ATCC, Manassas, Va., strain14468) from cultivated mycobacterial biomass was used to prepare MsRNC.Mycobacterium smegmatis cells were prepared as described in Example 1.Intact mycobacteria (Mycobacterium smegmatis) were first washed bylow-speed centrifugation to remove culture medium components, and werethen disrupted using high-pressure homogenization with an AvestinEmulsiFlex-05 high-pressure homogenizer followed by differentialcentrifugation as described in for MpRNC. In detail, a 10% w/vMycobacterium smegmatis cell suspension in water for injection wassubjected to 5 cycles of high-pressure homogenization at 18,000 psi. Thehomogenate was then centrifuged at 3,160×g for 30 min at 4° C. tosediment intact and undisrupted mycobacterium. The supernatant,containing RNA of the mycobacterium associated with mycobacterial cellwall fragments, was then centrifuged at 38,400×g for 1 hour at 4° C. toremove soluble contaminants and to pellet MsRNC. The MsRNC pellet wasthen re-suspended in water for injection and was homogenized for twoadditional cycles as described above. The suspension containingMycobacterium smegmatis RNC (MsRNC) was heat-treated at 121° C. forbetween 5-30 min.

Mycobacterium vaccae (strain ATCC15483, American Typing CultureCollections, Manassas, Va., USA) was cultivated in Middlebrook 7H9 brothsupplied with OADC enrichment (BD Diagnostic System, Sparks, Md., USA).The pelleted bacteria were stored at −20° C. prior to use). The use ofMiddlebrook 7H9 broth supplied with OADC enrichment (BD) allowed optimalgrowth of Mycobacterium vaccae as compared to the growth in thesynthetic medium due to the presence of as yet unknown growth factor(s)in the OADC enrichment. In this example, use was made of the newmanufacturing method described herein to ensure no additional exposureto exogenous materials. The cells were first washed by low-speedcentrifugation to remove culture medium components, and were thendisrupted using high-pressure homogenization with an AvestinEmulsiFlex-C5 high-pressure homogenizer followed by differentialcentrifugation as described for MpRNC. In detail, a 10% w/vMycobacterium vaccae suspension in water for injection was subjected to5 passages of high-pressure homogenization at 18,000 psi. Thesupernatant, containing RNA of the mycobacterium associated withmycobacterial cell wall fragments, was then centrifuged at 38,400×g for1 hour to remove soluble contaminants and to pellet MvRNC. The pelletwas then re-suspended in water for injection and was homogenized for twoadditional cycles as described above. The suspension containingMycobacterium vaccae RNC (MvRNC) was heat-treated at 121° C. for between5-30 min.

Example 5 Preparation of BRNC from Gram-Negative Bacteria

The BRNC compositions of the present invention can be prepared from oneor several Gram negative bacteria by for example disruption of thebacterium followed by phenol/chloroform/isoamyl alcohol extraction andethanol precipitation (as described in Short Protocols in MolecularBiology, 3rd Edition, Ausubel et al. Eds., John Wiley & Sons Inc., NewYork, USA). Use may be made of enzymatic procedures that are designed todigest unwanted chemical species prior to extraction of nucleicacid-containing compositions and thus optimize the yield. For example,Gram-negative bacteria were suspended in 5 mL RNase-free 50 mM Tris-HCl,5 mM EDTA, pH 8.0, adding RNase-free lysozyme (Sigma-Aldrich) to aconcentration of 1 mg/mL and incubating for 90 min at 37° C. RNase-freeproteinase K (Invitrogen, Burlington, Ontario, Canada) was then added togive a final concentration of 0.1 mg/mL, RNase-free sodium dodecylsulfate (BioRad, Richmond, Calif.) was added to give a finalconcentration of 1% w/v, and the incubation continued for 10 min at 65°C. Nucleic acid is then isolated by phenol/chloroform/isoamyl alcoholextraction. RNA-containing compositions can optionally be treated (forexample, by the use of high-pressure homogenization, mechanicalshearing, sonication, or autoclaving) to generate theoligoribonucleotides and polyribonucleotides of the present invention.

The bacterial RNA can optionally be used to prepare bacterial cell wallRNC formulations (BRNC), by the use of high-pressure homogenization anddifferential centrifugation and heat treatment as described in Examples1, 2, 3 and 4 of the present invention, such that they have anticancerand immune stimulant activity. The BRNC can be further combined withpharmaceutical carriers such as but not limited to cationic liposomes ornanoparticles as described in Examples 36 and 37.

Example 6 Preparation of BRNC or MRNC from Gram-Positive Bacteria

The BRNC compositions and the MRNC compositions of the present inventioncan be prepared from one or several Gram positive bacteria (includingmycobacteria) by, for example, disruption of the bacterium ormycobacterium followed by phenol/chloroform/isoamyl alcohol extractionand ethanol precipitation (as described in Short Protocols in MolecularBiology, 3rd Edition, Ausubel et al. Eds., John Wiley & Sons Inc., NewYork, USA). Use may be made of enzymatic procedures that are designed todigest unwanted chemical species prior to extraction of RNA and thusoptimize the yield. As an example of the applicability of the novelprocedures described above. Gram-positive bacteria are suspended in 5 mLRNase-free 50 mM Tris-HCl, 5 mM EDTA, pH 8.0, adding RNase-free lysozyme(Sigma-Aldrich) to a concentration of 1 mg/mL and incubating for 90 minat 37° C. RNase-free proteinase K (prepared from Tritirachium album.Invitrogen, Burlington, Ontario, Canada) is then added to give a finalconcentration of 0.1 mg/mL, RNase-free sodium dodecyl sulfate (BioRad,Mississauga, Ontario, Canada) is added to give a final concentration of1% w/v, and the incubation continued for 10 min at 65° C. RNA is thenisolated by phenol/chloroform/isoamyl alcohol extraction and ethanolprecipitation

The BRNC and MRNC can optionally be treated (for example, by the use ofhigh-pressure homogenization, mechanical shearing, sonication, or heattreatment) to generate the oligoribonucleotides and polyribonucleotidesof the present invention.

The bacterial RNA can optionally be used to prepare BRNC formulations,by the use of high-pressure homogenization and differentialcentrifugation and heat treatment as described in Examples 1, 2, 3 and 4of the present invention, such that they have anticancer and immunestimulant activity. The bacterial RNC can be further combined withpharmaceutical carriers such as but not limited to cationic liposomes ornanoparticles as described in Examples 36 and 37.

The mycobacterial RNA can optionally be used to prepare MRNCformulations, by the use of high-pressure homogenization anddifferential centrifugation and heat treatment as described in Examples1, 2, 3 and 4 of the present invention, such that they have anticancerand immune stimulant activity. The MRNC can be further combined withpharmaceutical carriers such as but not limited to cationic liposomes ornanoparticles as described in Examples 36 and 37.

Example 7 Morphological Examination of Mycobacterial Cell WallCompositions for Intact Mycobacterial Cells

A morphological comparison of mycobacterial cell wall compositions wascarried out using transmission electron microscopy (TEM). It is to berealized that in this example MpRNC is used as an illustrative andrepresentative example of the intact cell content of BRNC and MRNC.

A sample of Mycobacterium phlei biomass was prepared as described inExample 1, a sample of MpRNC High, Intermediate and Low was prepared asdescribed in Example 3, a sample of MCC was prepared as described inU.S. Pat. No. 6,326,357, a sample of MCWE was prepared as described inU.S. Pat. No. 5,759,554. Both of the two latter methods of manufacturinguse procedures that utilize PRONASE (Streptomyces griseusprotease)/trypsin digestion and phenol/urea treatment. All samples wereexamined morphologically using TEM. Samples prepared as a suspension inwater for injection (1 mg/mL w/v) were prefixed with 5% w/vglutaraldehyde (volume ratio: 1:1) for 1 hour. Following centrifugation(8,000×g for 10 min) the pellets were suspended in fresh 2.5% v/vglutaraldehyde and incubated overnight at 4° C. After incubation, thesamples were washed with washing buffer and kept in washing buffer at 4°C. prior to further processing. After washing three times in washingbuffer, the samples were pre-stained with osmium tetroxide and potassiumferrocyanide for 2 hours, washed, and dehydrated in acetone. Sampleswere then infiltrated with increasing concentrations of EPON™ (epoxyresin) in acetone, and polymerized at 58° C. for 48 hours. Thicksections (90-100 nm) were placed on 200-mesh copper grids, and stainedwith uranyl acetate followed by Reynolds lead. Samples were examined ineither a JEOL JEM-2011 200 kV w/Fas TEM w/Quartz XOne MicroanalyticalSystem and a Gatan DualView 300 W 1.3k×1k CCD Camera or a Philips CM200200 kV TEM equipped with an AMT 2k×2k CCD camera.

The results of the morphologic analysis are shown in FIG. 1. FIG. 1 ashows intact Mycobacterium phlei (used as a reference material), FIG. 1b shows MCC prepared according to U.S. Pat. No. 6,326,357, and FIG. 1 cshows MCWE prepared according to U.S. Pat. No. 5,759,554. Undisrupted M.phlei cells were observed as an electron dense structure, and containedvery few if any cell membrane or cell wall fragments. Unexpectedly,given the differential centrifugation step used in the preparation ofMCWE and MCC it was observed that their compositions were a mixture ofelectron dense and electron transparent structures consistent with amixture of intact mycobacteria and cell wall fragments (estimated to be8.5 and 3.0% of intact bacteria respectively). FIGS. 1 d, 1 e and 1 fshow that the number of intact mycobacteria in MpRNC low, medium andhigh respectively is controlled through the use of the manufacturingprocedures in Example 3 of the present invention (estimated to be 0.0,2.5 and 12.5% respectively).

The results of this morphological analysis demonstrate that themanufacturing process of the present invention results in a controllableintact mycobacterial cell content of MpRNC formulations.

Example 8 Manufacturing Contaminants in Mycobacterial Cell WallCompositions

Mycobacterial cell walls manufacturing according to U.S. Pat. No.5,759,554 (MCWE) and U.S. Pat. No. 6,326,357 (MCC) both use proteolyticenzyme digestion (Streptomyces griseus protease PRONASE/bovine trypsin)in conjunction with a phenol/urea treatment step. Such a procedure couldresult in proteolytic enzyme and phenol contamination of the final cellwall composition, even though both of these patents teach the use ofwashing procedures to eliminate these and other contaminants.

In this example the phenol content/contamination of mycobacterial cellwalls manufactured according to U.S. Pat. No. 5,759,554, U.S. Pat. No.6,326,357, and MRNC manufactured according to Example 3 of the presentinvention was determined by HPLC. MpRNC intermediate was used as arepresentative example of MRNC, and it is to be realized that themanufacturing principles taught in Example 3 of the present inventionare applicable to all MRNC as well as to BRNC.

Suspensions of the different mycobacterial cell wall compositions (1mg/mL in water for injection) or appropriate phenol standards in water(0-10 μg/mL) were treated with 12 M HCl at 100° C. for 60 min. Aftercooling, the mycobacterial cell wall compositions or phenol standardswere extracted with diethyl ether (2 mL diethyl ether/mg cell wallcomposition). The diethyl ether phase was removed and mixed with 0.05 MNaOH in methanol (3 mL/mg cell wall composition). After evaporation todryness using a stream of nitrogen, the residue was dissolved in water(0.2 mL/mg mycobacterial cell wall), and 100 μL was analyzed by HPLC forphenol content. The HPLC system for analysis utilized a Waters BreezeHPLC system consisting of a 717 plus auto sampler, a Waters 1525 BinaryHPLC Pump and a Waters 248L Dual wavelength absorbance detectorconnected to a C-18 reverse phase analytical 4.6×150 mm column (Waters,Nova-Pak C18, WAT 044375), packed with 4 μm silica (Waters Ltd.,Lachine, Quebec, Canada). HPLC separations were performed at roomtemperature using 100 μL sample volumes and at a flow-rate of 1.0 mL/minusing the following gradient elution conditions: Eluent A was wateracidified with 1% acetic acid (v/v) and Eluent B was 100% acetonitrile.Eluent A was maintained at 95% for 2 min and then decreased linearly to60% over 15 min, where it was held for 3 min. UV detection of phenol wasperformed at 270 nm.

For the detection of exogenous proteins, 20 mL of the differentmycobacterial cell wall compositions were lyophilized for a period of 72h. Proteins were extracted from the lyophilized mycobacterial cell wallcompositions using a modified RIPA extraction buffer (1% Triton X-100,0.5% NP-40%, 1% SDS, 150 mM NaCl, 1 mM EDTA, 10 mM Tris pH 7.5 at 4°C.). The total protein content of the extract was measured according tothe Macart method (Macart and Gerbaut, 1982 Clin. Chim. Acta122:93-101). Samples were diluted with Laemmli sample buffer (62.5 mMTris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue and 5%mercaptoethanol) to give a concentration of 1 mg of protein per mL. Atotal of 60 μL of diluted samples were loaded onto 10%-20% gradientSDS-PAGE gels (Bio-Rad, Mississauga, Ontario, Canada) andelectrophoresis was carried out at room temperature at 140V for 6 h to 8h hours. For calibration, molecular-weight standard mixtures (Bio-Rad,Mississauga, Ontario, Canada) were run in parallel with the samples.Gels were stained for protein using colloidal blue staining (Invitrogen,Carlsbad, Calif., USA) for 38 h at room temperature. Bands of interest(80, containing proteins of differing molecular weights) were excisedfrom the gels and placed in 96-well plates and then washed with water.Tryptic digestion was performed on a MassPrep liquid handling robot(Waters, Milford, Mass., USA) according to the manufacturer'sspecifications. Briefly, proteins were reduced with 10 mM DTT andalkylated with 55 mM iodoacetamide. Trypsin digestion was performedusing 105 mM modified porcine trypsin (Sequencing grade, Promega,Madison, Wis., USA) at 58° C. for 1 h. Digestion products were extractedusing 1% formic acid, 2% acetonitrile followed by 1% formic acid, 50%acetonitrile. The recovered extracts were pooled, vacuum centrifugedried and then suspended into 8 μL of 0.1% formic acid and 4 μL wereanalyzed by mass spectrometry. Peptide samples were separated by onlinereversed-phase (RP) nanoscale capillary liquid chromatography (nanoLC)and analyzed by electrospray mass spectrometry (ES MS/MS). Theexperiments were performed with a Thermo Surveyor MS pump connected to aLTQ linear ion trap mass spectrometer (ThermoFisher, San Jose, Calif.,USA) equipped with a nanoelectrospray ion source (ThermoFisher). Peptideseparation took place on a PicoFrit column BioBasic C18, 10 cm×0.075 mminternal diameter, (New Objective, Woburn, Mass.) with a linear gradientfrom 2-50% solvent B (acetonitrile, 0.1% formic acid) in 30 min, at 200nL/min (obtained by flow-splitting). Mass spectra were acquired using adata dependent acquisition mode using Xcalibur software version 2.0.Each full scan mass spectrum (400 to 2000 m/z) was followed bycollision-induced dissociation of the seven most intense ions. Thedynamic exclusion (30 second exclusion duration) function was enabled,and the relative collisional fragmentation energy was set to 35%. AllMS/MS samples were analyzed using Mascot (Matrix Science, London, UK;version 2.2.0). Mascot was set up to search the Uniref100 databaseassuming the digestion enzyme was trypsin. Mascot was searched with afragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 2.0Da. Iodoacetamide derivative of cysteine was specified as a fixedmodification and oxidation of methionine was specified as a variablemodification. Two missed tryptic digestion cleavage sites were allowed.Scaffold (Proteome Software Inc., Portland, Oreg.) was used to validateMS/MS based peptide and protein identifications. Peptide identificationswere accepted if they could be established at greater than 95.0%probability as specified by the Peptide Prophet algorithm (Keller, A etal Anal. Chem. 2002; 74(20):5383-92). Protein identifications wereaccepted if they could be established at greater than 95.0% probabilityand contained at least 2 identified peptides. Protein probabilities wereassigned by the Protein Prophet algorithm (Nesvizhskii, AI Anal Chem.2003 Sep. 1; 75(17):4646-58). Proteins that contained similar peptidesand could not be differentiated based on MS/MS analysis alone weregrouped to generate the least number of proteins.

The results obtained (Table 8) show that both MCWE and MCC arecontaminated with phenol and the protease aminopeptidase, a majorproteolytic enzyme found in the PRONASE of Streptomyces griseus. MpRNCmanufactured as described in Example 3, Table 7, did not contain phenolor any detectable PRONASE (Streptomyces griseus protease) components.

TABLE 8 Phenol, PRONASE (Streptomyces griseus protease) and trypsincontamination of mycobacterial cell wall compositions Streptomycesgriseus Phenol, ppm PRONASE Mycobacterial cell wall (ng/mg (aminoptidaseprotein) composition cell wall) (aminopeptidase)* MCWE U.S. Pat. No.5,759,554 652.53 YES MCC U.S. Pat. No. 6,326,357 657.89 YES MpRNCExample 3, present 0.00 NO invention

The data in Table 8 demonstrate that the use of phenol in themanufacture of mycobacterial cell walls leads to a detectable phenol andexogenous protein (PRONASE (Streptomyces griseus protease)aminopeptidase) contamination that in the case of MCWE and MCC wascomparable. One advantage of using the manufacturing procedures of thepresent invention whereby only RNase-free water for injection is used inobtaining MRNC compositions is that the resulting compositions are freeof phenol and exogenous protein contamination.

Phenol is amphiphilic and its subsequent presence in the mycobacterialcell walls is consistent with localization in the hydrophobic regions ofthe mycobacterial cell wall (covalently linked mycolic acids), where itremains trapped in spite of repeated centrifugation-based washes.

Analysis of contaminating proteins in MCWE and MCC, specificallyStreptomyces griseus aminopeptidase, were positive, thus confirming thatthe enzymatic treatment used in the preparation of mycobacterial cellwalls results in the presence of exogenous and potentially immunogenicproteins.

Example 9 Nucleic Acid Content and Profile of MpRNC CompositionsDetermined by Electrophoresis

MpRNC intermediate was used as a representative example of MRNC, and itis to be realized that analyses taught in this example are applicable toall MRNC and BRNC compositions.

MpRNC Intermediate suspension in water for injection collected beforeand after heat treatment (121° C., 30 min) was analyzedelectrophoretically for its nucleic acid profile using the Bioanalyzersystem (Bioanalyzer model #2100, Agilent, Santa Clara, Calif., USA). Thenucleic acid fraction was obtained as described in Example 6. Thenucleic acid fraction was diluted to a concentration of 30 ng/μL.Electrophoretic analysis of the length of the extracted nucleic acid wasaccomplished with the Bioanalyzer electrophoresis unit using the RNA6000 nano kit (Agilent #5067-1511). This kit provides information on thequality of RNA ranged from 25 to 6000 nucleotides. MpRNC Intermediatefollowing autoclaving was then further analyzed using the Small RNA Kit(Agilent Technologies Canada Inc., St. Laurent, Québec, Canada, kit#5067-1548), which is designed for the analysis of small nucleic acidsin the size range of 6 to 150 nucleotides. MpRNC High and MpRNC Low werealso analyzed after heat treatment (121° C., 30 min) using the Small RNAkit (Agilent). The results of the different electrophoretic analyses areshown in FIG. 2 a and FIG. 2 b.

FIG. 2 a demonstrates that MpRNC Intermediate prior to autoclavingpossesses a nucleic acid profile of between approximately 25 bases andclose to 4000 bases when analyzed using the RNA nano 6000 kit. Theresult demonstrates that the use of high-pressure homogenization steps(different pressurization and number of cycles) to prepare MpRNCIntermediate results in a composition that contains a polyribonucleotidechain length of 25-4000 bases. Following autoclaving, MpRNC Intermediateshows a more compact nucleic distribution ranged between 25 bases and200 bases (FIG. 2 b). Further analysis using the Small RNA Kitdemonstrated, in contrast to the profile obtained from the preparationprior to autoclaving, that all MpRNC preparations (High, Intermediateand Low) possess a nucleic acid profile that is comparable (FIG. 2 b)but that differs in quantity (High>Intermediate>Low).

FIG. 2 b demonstrates that MpRNC Intermediate possess anoligoribonucleotide peak that is maximal between 20-40 bases, and with anucleic acid profile of between 5 and 60 bases in length. The MpRNCcompositions had only minor amounts of nucleic acid material eluting at100 bases, and even less oligoribonucleotide material eluting at about150 bases in length. The results demonstrate that the use ofhigh-pressure homogenization and heat treatment steps (differentpressurization and number of cycles along with heat treatment) toprepare MpRNC results in a composition that contains anoligoribonucleotide and polyribonucleotide chain length of less than 60bases. Comparable nucleic acid profiles are also observed in MpRNC Lowand MpRNC High (FIG. 2 b).

Example 10 Analysis of Nucleic Acids in Mycobacterium phlei and MpRNCCompositions

In this example the nucleic acid content of Mycobacterium phlei andMpRNC compositions (high-, low- and intermediate-) were determined.MpRNC compositions were prepared as described in Example 3. It is to berealized that in this example MpRNC is used as an illustrative andrepresentative example of the nucleic acid content of MRNC.

Nucleic acids were extracted from the respective compositions using thefollowing procedure. An aliquot of the respective composition (700 μL ata concentration of 1 mg/ml) was digested with DNase- and RNase-freelysozyme, followed by inactivation and further digestion with DNase- andRNase-free proteinase K (both from Sigma-Aldrich Canada, Oakville,Ontario). Nucleic acids were extracted by phenol/chloroform/isoamylalcohol (25:24:1 v/v), and precipitated by the addition of glycogen,sodium acetate and ethanol. The precipitates were washed with 80%ice-cold ethanol, and solubilized in 50 μL distilled water. Theconcentration was determined by measurement of the absorbance at 260/280nm in a UV spectrophotometer. The nucleic acid content of eachcomposition is shown in Table 9.

TABLE 9 Nucleic acid content of MpRNC Composition Nucleic acid content,ng/mg Mycobacterium phlei 44,786 MpRNC Low 2,683 MpRNC Intermediate4,470 MpRNC High 15,686

These results demonstrate that an increasing amount of intactmycobacteria cells in MpRNC (e.g. MpRNC high) is associated with anincreasing amount of nucleic acids in the MpRNC composition. The resultsalso show that the nucleic acid content of MpRNC (2,683-15,686 ng/mg) issignificantly different to that of intact Mycobacterium phlei (44,786ng/mg).

Example 11 Determination of the DNA to RNA Ratio in MpRNC and AutoclavedMycobacterium phlei

MpRNC compositions were used as representative examples of MRNC, and itis to be realized that analyses taught in this example are applicable toall MRNC and BRNC compositions.

Analysis of the DNA to RNA ratio of MpRNC High, MpRNC Intermediate andMpRNC Low prepared as in Example 3 as well as autoclaved Mycobacteriumphlei cells (prepared as described in Example 1) was first performedusing enzymatic digestion with RNase-A of the extracted nucleic acidsfollowed by Bioanalyzer 2100 electrophoresis profiling andoligoribonucleotide content quantification by using the Agilent SmallRNA Kit (kit #5067-1548).

RNase-A digestion of the extracted nucleic acids (1260 ng/μL in RNasebuffer) was carried out using DNase-free Ribonuclease A (RNAse-A,treated at 100° C. for 30 min to remove DNase activity) (0.1 μg enzyme,2 h at 37° C.). The RNAse-A was obtained from Ameresco (Solon, Ohio,USA). A sample (20 ng/μL) of RNase A-treated nucleic acid was analyzedusing the Bioanalyzer. The amount of DNA and RNA was determined in MpRNCHigh, MpRNC Intermediate and MpRNC Low as well as intact autoclavedMycobacterium phlei cells using the equation:

DNA content=(Total Nucleic Acid Content−Nucleic Acid Content afterRNase-A treatment).

The results of the Bioanalyzer analysis are shown in FIG. 3 a for MpRNCHigh, FIG. 3 b for MpRNC Intermediate, 3 c for MpRNC Low and 3 d forautoclaved Mycobacterium phlei whole cells. The results of the analysisof MpRNC (high-, low- and intermediate-) with respect to RNA and DNAcontent are shown in Table 10.

TABLE 10 DNA and RNA content of MpRNC compositions DNA content RNAcontent Composition (% total nucleic acid) (% total nucleic acid MpRNCLow 0 100 MpRNC Intermediate 0.24 99.76 MpRNC High 3.6 96.4 Autoclaved4.0 96.0 Mycobacterium phlei

Second, analysis of the nucleic acid content and DNA to RNA ratio of theMpRNC's of the present invention (Low, Intermediate and High) wasperformed using a more sensitive quantitative methods as follows: Thenucleic acid fraction obtained in Example 6 was recovered byultrafiltration using a Microsep 1K unit (molecular weight cutoff=1000Da, Pall® Life Sciences, Ann Arbor, Mich., USA). The nucleic acidsolution was then digested to a mixture of nucleoside 5′-monophosphatesusing nuclease P1 (Sigma-Aldrich, Oakville, ON, Canada) following theprocedure reported by Liang (Liang et al., Ann. Chim. Acta 2009,650:106-110). To ensure optimal nuclease P1 digestion, a total of 50 μLof the nucleic acid aqueous solutions to be investigated were heated ina water bath at 95-100° C. for 10 min, followed by immediate chilling onice. Nuclease P1 was prepared at 5 units/μL in 30 mM sodium acetatebuffer containing 0.5 mM ZnCl₂, pH 5.3. For enzymatic digestion, 50 μLof nucleic acid in aqueous solution was mixed with the same volume ofnuclease P1 solution and then incubated at 50° ° C. for 30 min. Theresulting mixture was cooled to room temperature and filtered through aNanosep 10K filter by centrifugation at 10,000 g for 20 min at roomtemperature prior to HPLC analysis.

Serial dilutions of a mixture of mononucleotide standards (deoxyribo-and ribonucleotides, Sigma-Aldrich) containing 100, 10, 5, 2.5, 1 ng/μLeach of the mononucleotide present in DNA and RNA were also treated tonuclease P1 treatment and filtered for use as standards in HPLCanalysis. The elution order of these nucleotides was confirmed bycomparing the retention time of individual nucleotides under the sameHPLC condition. HPLC analysis was performed using a 1200 series HPLCsystem (Agilent, St-Laurent, Quebec, Canada), which was equipped with aquaternary pump with degasser, an auto sampler, a column heater, and amulti-wavelength UV detector. A ZORBAX Bonus-RP (reverse phase) column(Agilent Technologies) was used and the mobile phases comprised a lineargradient of 10 mM potassium phosphate buffer, pH 7.2 and methanol (0-10%methanol). The mononucleotides were detected at 260 nm.

The DNA:RNA ratio was determined after quantification of DNA and RNAdeoxyribonucleotides and ribonucleotides (Table 11).

TABLE 11 Quantity and DNA/RNA ratio in MpRNC High, MpRNC Intermediateand MpRNC Low DNA RNA Total DNA:RNA Sample ng/mg ng/mg ng/mg ratio MpRNCHigh 969.9 1241.3 2211.2 0.78 MpRNC Intermediate 907.1 542.6 1449.7 1.67MpRNC Low 649.7 309.5 959.2 2.10

These results demonstrate that in going from MpRNC high to MpRNC lowthere is an overall decrease in the nucleic acid content of ˜50%,consistent with a reduced intact Mycobacterium phlei cell content. AllMpRNC compositions in this analysis possessed both DNA and RNA, with theratio in MpRNC intermediate and MpRNC low being comparable. These dataare consistent with whole mycobacterial cell nucleic acid being removedin going from MpRNC high to MpRNC low, such that RNA in MpRNC is cellwall-bound and therefore optimally active with respect to biologicalactivity.

Example 12 RNA and DNA Proportions of MpRNC, MbRNC, MsRNC and MvRNC

Mycobacterial RNC compositions were analyzed electrophoretically fortheir RNA:DNA ratio using denaturing polyacrylamide gel electrophoresis(PAGE). It is to be understood that the mycobacteria used in thisexample are used as representatives for mycobacteria in general. MRNCfrom Mycobacterium phlei, Mycobacterium bovis strain BCG, Mycobacteriumsmegmatis and Mycobacterium vaccae was prepared as described in Examples3 and 4. The nucleic acid fraction was prepared as described in Example6. The nucleic acid fraction (50 μL) was divided into two aliquots of 25μL each. One aliquot was treated with 10 μg of RNase A (Amresco, Solon,Ohio, USA) and the other was treated with water for injection (Wisent,RNase and DNase-free) at 37° C. for 2 hours before loading onto apre-run denatured urea-PAGE gel at 25V/cm for 2.5 hour. The gel wasstained with SYBRGold® solution (Invitrogen) diluted 10,000× in 1× Trisborate buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0) for 15min before being visualized by UV at a wavelength of 312 nm. The gelimage was digitized using ImageJ image processing and analysis softwarefrom the NIH (Bethesda, Md., USA). The results are shown in FIG. 4. Thesize distribution of the nucleic acids demonstrated by denaturing PAGEelectrophoresis (FIG. 4 a) was confirmed by microfluidic chip RNAanalysis as described in example 11 for Mycobacterium phlei RNC.

The proportion of RNase-susceptible RNA is shown in FIG. 4 b. Thetreatment of the RNA fraction of the mycobacterial RNCs with RNase Aresulted in a 26.3% reduction in signal intensity for MbRNC, in a 46.1%reduction for MsRNC, in a 53% reduction for MpRNC, and a 60% reductionfor Mycobacterium smegmatis RNC. The MRNC compositions of the presentinvention therefore contain RNAse-susceptible RNA.

The higher proportion of RNA in Mycobacterium vaccae RNC, Mycobacteriumphlei RNC and Mycobacterium smegmatis RNC when compared to Mycobacteriumbovis BCG RNC is consistent with the fact that Mycobacterium phlei,Mycobacterium smegmatis and Mycobacterium vaccae are all rapidly growingspecies when compared to Mycobacterium bovis BCG, and that they have astronger ribosomal RNA expression promoter and an additional ribosomalRNA operon (Gonzalez-Y-Merchand, et al. 1997. J. Bacteriol. 179(22):6949-6958). Such data are consistent with an increased metabolicrequirement, expressed as increased RNA levels in both the mRNA and rRNApools of the faster-growing mycobacterial species.

Example 13 Lipid Content of MpRNC

The lipid content of and the molecular classes present in MpRNC weredetermined. It is to be realized that in this example MpRNC Intermediateis used as an illustrative and representative example of the lipidcontent of MRNC manufactured using the procedures described in Examples2 and 3 of the present invention. It also to be realized that differentmycobacterial species have different mycolic acid profiles, but that theprinciples taught in this example are applicable to the familymycobacteriaceae and genus mycobacterium.

Cell wall compositions from mycobacteria are often treated in theirpreparation with solvents to remove lipids (see for example Ribi et al.,1966, J. Bacteriol., 91:975-983, where the use of petroleum ether oracetone is described). Such procedures carry the risk of solventcontamination due to solubilization of the chemicals in covalentlyattached and non-extractable lipid species. Lipids of mycobacteria areconventionally divided into 2 groups: those that are non-covalentlybound, and that are therefore organic solvent or detergent extractable(including trehalose mono- and dimycolates and lipoproteins), and thosethat are covalently bound to the cell wall (mycolic acids). Unlesssaponification is performed, organic solvent or detergent extractiononly removes non-covalently bound lipids. The impact of themanufacturing process for MpRNC described in Example 3 of the presentinvention to on the lipid content of MpRNC was determined.

A modification of the Dole procedure for extraction of non-covalentlybound free lipids, including phospholipids, glycolipids and free fattyacids was used (Dole and Meinertz, 1960, J. Biol. Chem. 235:2595-2599,as modified by Puttmann et al., 1993 Clin. Chem. 39:825-832). One mL ofMpRNC Intermediate pellet or supernatant from step 3 of themanufacturing process in Example 3 prepared from a 10 mg/mL MpRNCIntermediate suspension in water for injection and subjected tohigh-speed centrifugation was added to 3 mL of the solvent mixturen-heptane/isopropanol/phosphoric acid (10/40/1, v/v/v) and mixedvigorously in a glass tube with a Teflon® lined cap. The mixture wasincubated at room temperature for 30 min. After adding 1 mL ofn-heptane, followed by 1 mL of water, the tube was capped tightly, mixedthoroughly and was left to settle. The upper organic phase was collectedand the remaining aqueous phase containing dilapidated cell wall orsupernatant was extracted by the addition of 1 mL of heptane. The twoheptane phases, containing extractable, non-covalently attached freelipids, were then pooled and dried at 50° C. under a stream of nitrogenin a glass tube with a Teflon® lined cap until thoroughly dried. The drylipid extracts were stored at 4° C. protected from light untilderivatization. The residue containing free lipid-depleted MpRNC wascollected, kept at −20° C. and used for the extraction of covalentlybound lipids (defined as the covalently linked mycolic acid cell wallfraction).

The derivatization of the fatty acids for HPLC was carried out asfollows. First, 0.1 mL of KHCO₃ solution (4 g KHCO₃ dissolved in 98 mLwater and 98 mL methanol) was added to dry free lipid extracts and airdried at 50° C. under stream of filtered air. Then 1 mL of n-heptane and50 μl of p-bromophenacyl-8 reagent (Pierce Chemical Co. Rockford, Ill.,USA) was added, and mixed thoroughly. The Tubes were sealed and heatedfor 30 min at 85° C. After cooling to room temperature, the solution wasclarified by adding 1 mL of an HCl (6 M)-methanol-water mixture (1:2:1),and vortexing vigorously. The tubes were left to stand for 5 min andthen the organic layer collected in a glass vial and dried under streamof nitrogen. The dry p-bromophenacyl derivatized free lipids were keptat 4° C. protected from light until reverse-phase-HPLC with UV detectionanalysis (RP-HPLC-UV).

RP-HPLC-UV analysis of p-bromophenacyl ester derivatives of thenon-covalently bound lipids and free fatty acids was carried out using aWaters Breeze HPLC system with a binary pump, and a dual wavelengthabsorbance UV detector (Waters Ltd., Lachine, Quebec, Canada). A 4micron reverse phase separation column (Waters, Nova-Pak, C18 4.6 mm×150mm) was used for analysis. The dried p-bromophenacyl-ester fatty acidderivatives were dissolved in 100 μL acetonitrile and 5 μL was injectedinto the column for separation using a gradient mixture of 77%acetonitrile and 23% water for 0 to 20 min: from 20 min to 25 min theconcentration of acetonitrile was raised from 77% to 96% and thendecreased from 96% to 77% for the last 5 min of analysis at a flow rateof 0.25 mL/min. The column temperature was at 30° C. and the peaks weredetected at 254 nm. Myristic acid (C14:0) (Sigma-Aldrich, Oakville,Ontario, Canada) was used as fatty acid standard for quantification offree lipids.

The fraction from the step 3 pellet and the step 3 supernatant remainingafter the extraction of non-covalently bound lipid was dried under astream of nitrogen at 50° C., then hydrolyzed with 2 mL hydrolysisreagent (50% w/v potassium hydroxide in water and 50% methanol, 1:1,v/v) for 1 hour at 100° C. in a glass tube with a Teflon® lined cap torelease covalently-bound lipid. After cooling to room temperature, 2 mLof chloroform was added followed by 1.5 mL acidification reagent(concentrated HCl/methanol (1:1, v/v)). After vigorous mixing the tubeswere left to stand for 5 min for phase separation. The chloroform phase(lower phase) was recovered and transferred into a new glass tube with aTeflon®-lined cap. The remaining aqueous phase was then re-extractedwith 1 mL chloroform 2 more times. The 3 chloroform phases were pooledand dried at 80° C. under a stream of nitrogen and kept at 4° C.protected from light until derivatization.

The derivatization of the hydrolyzed covalently attached fatty acids wascarried out as described for the extractable lipids except chloroformwas used instead of n-Heptane. Dry p-bromophenacyl derivatized fattyacids were kept at 4° C. protected from light until RP-HPLC-UV freelipid analysis.

The RP-HPLC-UV analysis p-bromophenacyl ester derivatives of thecovalently attached lipids (defined as the mycolic acid fraction in thisanalysis) was carried out using a Waters Breeze HPLC system with abinary pump, and a dual wavelength absorbance UV detector. A 4 micronreverse phase separation column (Waters, Nova-Pak, C18 4.6 mm×150 mm)was used for separation. The dried p-bromophenacyl ester mycolic acidderivatives were dissolved in 100 μL dichloromethane, 20 μL injectedinto the column and detection was at 254 nm. Behenic acid (C22:0,Sigma-Aldrich) was used as an internal standard for quantification ofcovalently bound lipids. The column was eluted with using amethanol/dichloromethane gradient (98% methanol/2% dichloromethane to20% methanol/80% dichloromethane over 20 min) at a flow rate of 2.5mL/min. Behenic acid (C22:00, Sigma-Aldrich, Oakville, Ontario, Canada)was used as internal standard for quantification, and internal low andhigh mycolic acid standards (generally accepted as being ˜C40:n and˜C110:n respectively, and originally supplied by the Corixa CorporationSeattle, Wash., USA) were used to estimate the carbon chain length ofthe mycolic acids in cluster 1 and cluster 2.

The results of the noncovalently-linked lipid (extractable) andcovalently-linked lipid analyses for MpRNC Intermediate are shown inFIG. 5 a and FIG. 5 b and in Table 5. FIG. 5A shows that the boundlipids after extraction and hydrolysis are comprised of a majority ofshort-chain fatty acids with a carbon chain length less than C22, andthat the 2 clusters of mycolic acid are present that are characteristicof Mycobacterium phlei. FIG. 5B shows that the hydrolysable lipidobtained after saponification is composed of both low and high molecularweight species, and that the 2 mycolic acid clusters fall between the 2mycolic acid standards (FIG. 5 b). The relationship between retentiontime and carbon chain length for the mycolic acid standards wasdetermined to be linear.

Lipids peaks obtained after saponification were classified asshort-chain fatty acids (defined in this analysis as the result of thebreakdown of mycolic acid wax esters during hydrolysis into lowermolecular weight dicarboxylic acids and an alcohol) with an elution timeof 0.5-5 min, mycolic acid cluster 1 (elution time 7-9 min, comprisingmycolic acids of between 45 and 55 carbons) and mycolic acid cluster 2(elution time 10.5-13.5 min comprising mycolic acids of between 70-85carbons). The amounts of each of these fractions in the step 3 pelletand supernatant are shown in Table 12

TABLE 12 Non-covalent and covalently-bound lipids in MpRNC IntermediateNon-extractable, covalently bound cell wall lipid (includingExtractable, mycolic acids), μg/mL non- Total Total Lipid, μg/mL) MpRNCcovalently mycolic acid Total prepared as bound lipid, Short (cluster1 + Total covalent + in Example 2 μg/mL chain cluster 2) covalentextractable MpRNC 15.4 104.4 31.1 135.5 151.0 (high speed centrifuga-tion pellet) MpRNC 307.4 22.1 1.7 23.7 331.2 high speed centrifuga- tion

The high-speed centrifugation pellet of MpRNC Intermediate from step 3of Example 3, containing MpRNC composition, contained extractable lipid,as well as covalently bound lipid. The covalently-bound lipid,considered to represented mycolic acids, represented 90% of the totallipid. The high-speed supernatant, in contrast, contained very littlecovalently bound lipid, with most of the lipid being non-covalentlybound (93%).

In another experiment, a total of 30 mg of MpRNC Intermediate and MCCwere dried under a stream of nitrogen at 50° C., then hydrolyzed with 2mL hydrolysis reagent (50% w/v potassium hydroxide in water and 50%methanol, 1:1, v/v) for 1 hour at 100° C. in a glass tube with a Teflon®lined cap to release covalently-bound lipid. After cooling to roomtemperature, 2 mL of chloroform was added followed by 1.5 mLacidification reagent (concentrated HCl/methanol (1:1, v/v)). Aftervigorous mixing the tubes were left to stand for 5 min for phaseseparation. The chloroform phase (lower phase) was recovered andtransferred into a new glass tube with a Teflon®-lined cap. Theremaining aqueous phase was then re-extracted with 1 mL chloroform 2more times. The 3 chloroform phases were pooled and dried at 80° C.under a stream of nitrogen and weighed. The extraction was carried outtwice and the lipid content of was 14.1±5.1 mg/30 mg MCC and 9.7±0.14mg/30 mg MpRNC Intermediate.

The presence of such a high lipid content (and low mycolic acid content)in the supernatant is consistent with the liberation of non-cell walllipids (extractable, non-covalently-bound fatty acids and non-covalentlylinked mycolates) following disruption of the mycobacteria by highpressure homogenization, and which are removed from the cell wallcomposition by the differential high-speed centrifugations stepsdescribed in Example 3. This procedure therefore eliminates thenecessity of organic solvent or detergent extraction and the risk ofcontamination as a means of reducing the lipid content of mycobacterialcell wall compositions. The covalently attached mycolic acids, anintegral component of mycobacterial cell walls, were however conservedby the manufacturing method of Example 3, thus providing the benefit ofdefinitive identification of cell walls from a given mycobacterialspecies by mycolic acid cluster analysis.

Example 14 Diaminopimelic Acid (DAP) and Alanine Content of MpRNC High,MpRNC Intermediate, MpRNC Low and of Autoclaved Mycobacterium phlei

In this example the MpRNC intermediate composition was used as arepresentative example of MRNC, and it is to be realized that analysestaught in this example are applicable to all MRNC, and many BRNCcompositions prepared from but not limited to Nocardia species,Rhodococcus species, and Corynebacteria species.

Diaminopimelic acid (2,6-diaminoheptanediotic acid, DAP) and alanine(ALA) are major constituents of the peptidoglycan of Mycobacteriumphlei. Peptidoglycan is itself a major component of mycobacterial cellwalls. DAP is involved in interpeptide chain linkages, and ALA is acomponent of the peptide chain of peptidoglycan. The measurement of DAPand ALA content thus provides an indirect determination of the degree ofintact mycobacterial cell content whereby increasing mycobacterial cellcontent results in a decreased DAP content. The DAP and ALA content ofMpRNC High, MpRNC Intermediate MpRNC Low and autoclaved Mycobacteriumphlei cells were determined by HPLC analysis. MpRNC High, MpRNCIntermediate, MpRNC Low and autoclaved Mycobacterium phlei cells werediluted in HPLC grade water to a concentration of 0.2 mg/mL. A 20 μLaliquot of each diluted sample preparation along with a respectivehydrolysis blank and bovine serum albumin control were dried undervacuum in pyrolysis tubes and hydrolyzed at 165° C. for 60 min using 6MHCl vapor. DAP and ALA were derivatized with6-aminoquiolyl-N-hydroysuccinimidyl carbamate (AQC) (AccQ Fluor Reagentkit, Waters, Milford, Mass., USA). Standard curves were prepared usingsynthetic and commercially available DAP and ALA. The DAP and ALAcontent of MpRNC High, MpRNC Intermediate, MpRNC Low and of autoclavedMycobacterium phlei cells was determined using triplicate samples byHPLC (5 μL injection volume of each sample using an ACCQ-Tag Novo-PakC₁₈ 4 μm, 3.9×150 mm column [Waters] and an Agilent Model 1100, SantaClara, Calif., USA HPLC equipped with fluorescence and UV detectors).The results of the DAP and ALA content determinations are shown in Table13.

TABLE 13 DAP and ALA content of MpRNC High, MpRNC Intermediate, MpRNCLow and autoclaved Mycobacterium phlei. Composition ALA (mol %)* DAP(mol %)* MpRNC High 15.8 2.6 MpRNC Intermediate 21.6 7.0 MpRNC Low 23.810.1 Autoclaved 14.8 2.7 Mycobacterium phlei *The results for ALA andDAP are expressed as the mol % of the amino acid analysis.

Table 13 demonstrates that with a decreasing number of intactmycobacterial cells present in MpRNC (High to Low), there is acorresponding increase in both the ALA and DAP content. The results areconsistent with an increase in cell wall peptidoglycan content in theMpRNC compositions in going from MpRNC High to MpRNC Low concomitantwith a decrease in intact mycobacterial cells. Table 11 and Table 13together also show that isolation of anoligoribonucleotide/polyribonucleotide formulation compositioncontaining cell walls is associated with increasing amounts ofpeptidoglycan (as determined by DAP and alanine measurements, or byalanine measurements (for those bacteria and mycobacteria that do notcontain DAP) and that decreasing amounts of DAP and alanine areassociated with increasing levels of intact mycobacteria. Suchmeasurements can therefore be used to accurately verify and control theintact mycobacterial (or bacterial) content of such compositions

Example 15 MpRNC Protein Content

In this example the MpRNC Intermediate composition was used as arepresentative example of MRNC, and it is to be realized that theanalyses taught in this example are applicable to all MRNC and BRNCcompositions.

The protein content of MpRNC Intermediate prepared as described inExample 3 of the present invention was compared with that ofMycobacterium phlei cells. To ensure an accurate determination ofprotein content the samples were prepared as 1 mg/mL suspension in waterfor injection and subjected to probe ultrasonication to ensuredisruption of any intact mycobacteria and thus ensure an accuratedetermination of protein. Protein content was determined before andafter sonication. M. phlei intact cells (grown according to Example 1 ofthe present invention using MCMC-C) and MpRNC Intermediate manufacturedaccording to Example 3 of the present invention (both at a concentrationof 1 mg/mL in water for injection) were subjected to probe sonication(Misonix Inc, ¼″ probe, setting 8, 1-15 min on ice). Samples wereremoved at various times for protein analysis using the Macart method.Briefly, the protein content was determined by mixing 15 μL ofappropriately Mycobacterium phlei or MpRNC Intermediate with 300 μL ofMACART solution (L. Gerbaut and M. Macart, Clinical Chemistry (1982) 32,353-355) in the wells of microtiter plates and incubating for 5 min atroom temperature, after which the OD was determined at 630 nm using amicroplate reader. The amount of protein in each sample was determinedusing as a reference serial dilution of bovine serum albumin (2 mg/mLProtein Standard, Sigma). The results of the analysis are shown in Table14.

TABLE 14 Protein content of Mycobacterium phlei and MpRNC IntermediateSonication time Protein content (μg/100 μg)* (min) Mycobacterium phleiMpRNC Intermediate 0 25 13 1 29 10 5 36 12 15  45 17 Fold change 1.8 1.3(0 min-15 min) *Mean protein content of a triplicate analysis.

The data shown in Table 14 show that MpRNC Intermediate manufacturedaccording to the method described in Example 3 had a significantlydifferent protein content when compared to Mycobacterium phlei (13% w/vversus 45% w/v respectively), thus demonstrating that the manufacturingprocess results in a decrease in protein content when compared with thestarting cell biomass. These data show also that without the use of anadditional disruption procedure, the protein content of MpRNC wasunderestimated by approximately 30%. The slight increase in proteinobtained after sonication of MpRNC Intermediate is consistent with thepresence of 3 protein-containing compartments-cell wall fragmentproteins accessible to the protein analysis reagent, proteins withincell wall fragments that are not accessible to the protein analysisreagent, and proteins within residual, intact mycobacteria, and are alsonot accessible to the protein analysis reagent. These proteins becomeaccessible following disruption of the cell wall fragments or cells bysonication or other means. It is to be understood that the protein levelof MpRNC Low and MpRNC Intermediate and MpRNC High will be a reflectionof the ratio of intact cells to cell wall fragments in the respectiveformulations.

Example 16 MpRNC Induces the Activation of the Human Pathogen-AssociatedMolecular Pattern Receptor (PAMP) NOD2

Nucleotide-binding oligomerization domain 2 (NOD2) is a patternrecognition receptor (PRR), one of a diverse family of receptorsresponsible for sensing pathogen-associated molecular patterns (PAMPs)associated with microbial pathogens or cellular stress. NOD2 is areceptor for muramyl dipeptide (MDP), known to be the minimal structureof bacterial peptidoglycan that possesses immune stimulant activity.NOD2 MDP agonists are known to activate innate immune system cells(monocytes, macrophages and dendritic cells), modulate inflammation,have analgesic activity, possess anticancer activity, possessanti-infection activity, induce the differentiation of human bone marrowCD34⁺ cells and increase vaccine protection through their immuneadjuvant activity. MDP and a large number of analogs and derivativeshave been shown to be effective agents for the treatment of cancer andinfection, and can act as vaccine adjuvants.

The NOD2 activating activity of MpRNC was evaluated using HEK-293 cellsengineered to express the human NOD2 receptor and a downstreamsignalling marker IL-8 under the control of NOD2-driven NF-κB. It is tobe understood that MpRNC is used as an example of MRNC, and that theprinciples taught in this Example are applicable to MRNC from themycobacteriaceae family. The ability of MpRNC Intermediate to activateNOD2 was compared with MCC prepared according to U.S. Pat. No.6,326,357.

The human HEK293-NOD2 cell line (InvivoGen, San Diego, Calif., USA) wascultured and maintained in high glucose DMEM, supplemented with 10%fetal bovine serum (both from Wisent, St-Bruno, Québec, Canada), 100μg/mL Normocin™ and 10 μg/mL blasticidin (both from InvivoGen) at 37° C.in a humidified atmosphere containing 5% CO₂. HEK293-NOD2 cells wereseeded at 5×10⁵ cells/mL in a volume of 0.2 mL in sterile 96-wellflat-bottomed tissue culture microplates in the cell culture mediumdescribed above, and incubated for 48 hours with 0.625, 1.25, 2.5, 5,10, 20, 40, 80 and 160 μg/mL MpRNC Intermediate or MCC at 37° C. in ahumidified atmosphere containing 5% CO₂. Supernatants were collectedafter incubation, centrifuged at 4,000×RCF for 5 min at 4° C. to removecells and debris and the supernatant was stored at −20° C. for analysis.Human IL-8 in the supernatant was measured after 48 hours cultivationwith MpRNC Intermediate suspension by means of a commercialenzyme-linked immunosorbent assay (ELISA) from BioSource, Camarillo,Calif., USA. Data were captured from an ELISA plate reader (ELx-808IUBioTek Instruments, Winooski, Vt., USA) using the KC Junior softwarepackage (BioTek Instruments, Winooski, Vt., USA) and expressed in pg/mLof IL-8 synthesized.

Table 15 shows that MpRNC Intermediate functions in a dose-relatedmanner as a potent NOD agonist by inducing NOD2-driven IL-8 release in aconcentration-dependent manner, while MCC prepared according to U.S.Pat. No. 6,326,357 was significantly less potent as a NOD agonist, withmarginal activity only being seen at the highest concentration tested.

TABLE 15 NOD2 activating activity of Mycobacterial cell wallcompositions IL-8 (pg/mL) Cell wall concentration, μg/mL Composition0.625 1.25 2.5 5.0 10 20 40 80 160 MpRNC Intermediate 45.1 42.7 51.778.3 110.0 124.7 133.2 160.1 186.9 MCC (U.S. Pat. No. 27.7 20.5 23.816.3 14.9 11.2 13.4 16.3 36.6 6,326,357)

It is clear that MpRNC has significant and unexpected advantages overother mycobacterial cell wall compositions such as MCC with respect toimmune stimulant activity mediated through the NOD2 receptor.

In a separate study the NOD2-activing activity of MpRNC High, MpRNCIntermediate and MpRNC Low was compared by determining the potency ofMpRNC High and MpRNC Low relative to that of MpRNC Intermediate.NOD2-activating activity was determined in the dose range 0.625, 1.25,2.5, 5, 10, 20, 40 and 80 μg/mL as described above, and relativepotencies were calculated using PHARM/PCS version 4.2 software(Microcomputer Specialists, Philadelphia, Pa., USA). The results shownin Table 16 demonstrate that the NOD2 activation activity of MpRNC Highand of MpRNC Low is not different to that of MpRNC Intermediate.

TABLE 16 The relative potencies of MpRNC (high, intermediate and low)for the activation of NOD2 MpRNC Relative potency MpRNC Intermediate 1.0MpRNC High 0.9 MpRNC Low 1.1

These data demonstrate that the presence of variable levels of intactmycobacteria in the different MpRNC compositions has no effect on theirability to activate NOD2, and that all MpRNC compositions (High,Intermediate and Low) possess the same ability to activate this innateimmune system receptor.

Example 17 Mycobacterial RNC Induce the Activation of the HumanPathogen-Associated Molecular Pattern Receptor (PAMP) NOD2

The NOD2 activation activity of mycobacterial RNC prepared fromMycobacterium bovis BCG, Mycobacterium smegmatis and Mycobacteriumvaccae was compared with RNC from Mycobacterium phlei usingHEK-Blue™-NOD2 cells engineered to express the human NOD2 receptor(InvivoGen, San Diego, Calif., USA) activation of which drives thesynthesis of secretory embryonic alkaline phosphate (SEAP) under thecontrol of NOD2-driven NF-κB. Measurement of SEAP in the supernatantprovides a measure of NOD2 activation.

It is to be understood that MRNC from Mycobacterium phlei, Mycobacteriumbovis BCG, Mycobacterium smegmatis and Mycobacterium vaccae are used asexamples representative of the mycobacteriaceae family, and that theprinciples taught and data shown in this Example are applicable to MRNCfrom other mycobacteria. The MRNC Intermediate composition fromMycobacterium phlei (MpRNC) were used in this example, given thedemonstration of comparability of activity between MpRNC High,Intermediate and Low in Example 16.

The HEK293-NOD2 cell line was cultured and maintained in high glucoseDMEM, supplemented with 10% fetal bovine serum (both from Wisent,St-Bruno, Québec, Canada), 30 μg/mL blasticidin, 100 μg/mL of Normocin™and Zeocin™ (both from InvivoGen) as well as 50 U/mL penicillin and 50μg/mL streptomycin (all from Wisent) at 37° C. in a humidifiedatmosphere containing 5% CO₂. The cells were seeded at 1×10⁵ cells/mL ina volume of 0.2 mL in sterile 96-well flat-bottomed tissue culturemicroplates in the cell culture medium described above, and incubatedfor 72 hours with 0.625, 1.25, 2.5, 5, 10, 20, 40, and 80 μg/mL of MRNCIntermediate. Supernatants were collected for analysis after incubation,and were centrifuged at 4,000×RCF for 5 min at 4° C. to remove cells anddebris. Twenty μL of supernatant was mixed with 180 μL of QUANTI-Blue™(InvivoGen) in a 96-well flat-bottomed microtiter plates at 37° C. for 2hours. SEAP expression was measured at 630 nm using an ELx-808IUmicroplate reader (BioTek Instrument, Winooski, Vt.) using the KC Juniorsoftware package (BioTek Instruments). FIG. 6 shows the dose-relatedactivation of NOD2 by all four MRNC compositions.

All MRNC compositions demonstrated NOD2 activating activity.Mycobacterium phlei RNC was the most effective activator of NOD2,followed by Mycobacterium smegmatis, Mycobacterium bovis BCG andMycobacterium vaccae. Both Mycobacterium bovis BCG and Mycobacteriumvaccae showed marginal activation of NOD2 at the highest dose tested (80μg/mL). The potency of the mycobacterial RNC compositions relative toMycobacterium phlei RNC for the activation of NOD2 was determined usingPharmPC V4.2 (Microcomputer Specialists, Philadelphia, Pa.) and is shownin Table 17.

TABLE 17 Mycobacterial RNC NOD2 activation relative to Mycobacteriumphlei MpRNC NOD2 potency relative to Mycobacterial RNC Mycobacteriumphlei RNC MpRNC intermediate 1.00 MbRNC intermediate 0.014 MsRNCintermediate 0.021 MvRNC intermediate 0.013

Example 18 MpRNC Induces the Activation of the Human Pathogen-AssociatedMolecular Pattern Receptor (PAMP) TLR2

Toll-like receptor 2 (TLR2) is a pattern recognition receptor (PRR)responsible for sensing pathogen-associated molecular patterns (PAMPs)commonly associated with microbial pathogens. Bacterial andmycobacterial cell wall structural components such as peptidoglycan,lipomannan (LM), lipoarabinomannan (LAM), lipoproteins and lipopeptidesappear to be largely responsible for the activation of TLR2. It is knownby those skilled in the art that TLR2 is expressed on many immune systemcells, including macrophages, and that agonists of TLR2 are capable ofinducing the synthesis of a number of chemokines and cytokines by suchcells. The TLR2 activation activity of MpRNC Intermediate prepared asdescribed in Example 3 of the present invention and MCC preparedaccording to U.S. Pat. No. 6,326,357 were compared using HEK293 cellsengineered to express the human TLR2 receptor, thus providing aconvenient and accepted method of determining immune stimulantpotential.

It is to be understood that MpRNC Intermediate is used as an example ofMRNC, and that the principles taught in this Example are applicable toMRNC from other mycobacteria, as well as to the mycobacteriaceae family.

The human HEK293-TLR2 cell line was obtained from InvivoGen, San Diego,Calif., USA. HEK293-TLR2 cells are stably transfected with the TLR2 geneand a receptor-driven secreted embryonic alkaline phosphatase gene(SEAP), placed under the control of the NF-kB gene. Activation of TLR2thus results in the generation of alkaline phosphate activity in thecell culture medium, which is used to quantify receptor activation. Thecells were cultured and maintained in high glucose DMEM, supplementedwith 10% fetal bovine serum (both from Wisent, St-Bruno, Québec, Canada)and 1× Normocin™ (InvivoGen) at 37° C. in a humidified atmospherecontaining 5% CO₂. HEK293-TLR2 cells were seeded at 5×10⁵ cells/mL in avolume of 0.2 mL in sterile 96-well flat-bottomed tissue culturemicroplates in HEK-Blue™ Detection medium (InvivoGen), and incubated for18 hours with 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 μg/mL MpRNCIntermediate or MCC at 37° C. in a humidified atmosphere containing 5%CO₂. The HEK-Blue™ Detection medium added to the wells is designed forthe detection of NF-kB induced soluble embryonic alkaline phosphataseenzyme activity (SEAP activity). After an 18 hour incubation, theoptical density (which is proportional to the activation of NF-kBthrough TLR2 engagement) was determined at 630 nm using a microplatereader (ELx-808IU model, BioTek Instrument, Winooski, Vt., USA). Datawas captured using the KC Junior software package (BioTek Instruments).

Table 18 shows the activation of TLR2 (expressed as the OD at 630 nm)and demonstrates that MpRNC Intermediate prepared using the proceduredescribed in Example 3 has dose-related TLR2 activating activity.Determination of the potency of MpRNC Intermediate relative to MCCshowed that although the activities were comparable to that of MCCprepared as described in U.S. Pat. No. 6,326,357. MpRNC Intermediate was10.6 fold more potent than MCC as a TLR2 activator (determined usingPHARM/PCS version 4.2 software, Microcomputer Specialists, Philadelphia,Pa., USA).

TABLE 18 TLR2 activating activity of MpRNC Intermediate and MCC TLR2activation (SEAP O.D.) Mycobacterial cell wall composition, μg/mLComposition 0 0.625 1.25 2.5 5.0 10 20 40 80 160 MpRNC 0.15 0.60 0.380.52 0.50 0.59 0.55 0.64 0.71 0.74 Intermediate MCC (U.S. Pat. 0.15 0.420.40 0.40 0.49 0.45 0.56 0.51 0.57 0.61 No. 6,326,357

These data in conjunction with the data from the evaluation of theability of MpRNC to activate NOD2 as shown in Example 16, demonstratethat MpRNC cell wall compositions possess dual agonist activity towards2 key receptors of the innate immune system, specifically the ability toactivate both NOD2 and TLR2 without resort to the use of syntheticagonists.

Example 19 Mycobacterial RNC Induces the Activation of the HumanPathogen-Associated Molecular Pattern Receptor (PAMP) TLR2

The TLR2 activating activity of the mycobacterial RNC compositions asprepared in Example 4 was determined using the HEK-293 cell reportersystem engineered to express the human TLR2 receptor and compared withMycobacterium phlei RNC Intermediate.

The human HEK293-TLR2 cell line was obtained from InvivoGen, San Diego,Calif., USA. The HEK293-TLR2 cells are stably transfected with the TLR2gene and a receptor-driven secreted embryonic alkaline phosphatase gene(SEAP), placed under the control of NF-kB. Activation of TLR2 thusresults in the generation of alkaline phosphate activity in the cellculture medium, which is used to quantify receptor activation. The cellswere cultured and maintained in high glucose DMEM, supplemented with 10%fetal bovine serum (both from Wisent, St-Bruno, Québec, Canada) and 1×Normocin™ (InvivoGen) at 37° C. in a humidified atmosphere containing 5%CO₂. HEK293-TLR2 cells were seeded at 5×10⁵ cells/mL in a volume of 0.2mL in sterile 96-well flat-bottomed tissue culture microplates inHEK-Blue™ Detection medium (InvivoGen), and incubated for 18 hours with2.5, 5, 10, 20, 40, and 80 μg/mL mycobacterial RNC at 37° C. in ahumidified atmosphere containing 5% CO₂. The HEK-Blue™ Detection mediumadded to the wells is designed for the detection of TLR2/NF-kB inducedsoluble embryonic alkaline phosphatase enzyme activity (SEAP activity).After an 18 hour incubation, the optical density (which is proportionalto the activation of NF-kB through TLR2 engagement) was determined at630 nm using a microplate reader (ELx-808IU model, BioTek Instrument,Winooski, Vt.). Data was captured using the KC Junior software package(BioTek Instruments).

FIG. 7 shows that all 4 mycobacterial RNC induced the activation of TLR2in a dose-related manner in the dose range 2.5-80 μg/mL. Mycobacteriumvaccae RNC was the most active, followed in decreasing order of activityby Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacteriumphlei RNC. The potency of the mycobacterial RNC compositions relative toMycobacterium phlei RNC for the activation of TLR2 was determined usingPharmPC v4.2 (Microcomputer Specialists, Philadelphia, Pa., USA) and isshown in Table 19.

TABLE 19 Mycobacterial RNC TLR2 activation relative to Mycobacteriumphlei MpRNC TLR2 activation potency relative to Mycobacterial RNCMycobacterium phlei RNC Intermediate MpRNC Intermediate 1.00 MbRNCIntermediate 1.56 MsRNC Intermediate 3.06 MvRNC Intermediate 5.86

The range of potencies observed for the activation of TLR2 (<6-folddifference range) indicate that all 4 mycobacterial RNC compositionshave comparable TLR2 agonist activity. It is clear that one skilled inthe art upon reading this and other examples in the present application(for example NOD2 activation) will make use of the most appropriatecombination of immune stimulant activities and immune system receptoragonist function associated with a particular mycobacterial RNCcomposition in determining the applicability of a specific mycobacterialRNC or combination thereof for a particular or specific prophylactic ortherapeutic application.

Example 20 Stimulation of TLRs by MpRNC Intermediate

TLR2 agonists are generally recognized as being associated with cellwall PAMPs from Gram positive bacteria and mycobacteria. TLR3 agonistsare generally recognized as double-stranded viral RNA. TLR4 agonists aregenerally recognized as being lipopolysaccharide. TLR5 interactsspecifically with bacterial flagellin. TLR7 and TLR8 agonists aregenerally recognized as being single-stranded RNA. TLR9 agonists aregenerally recognized as being unmethylated DNA containing CpGdinucleotide sequences (CpG motif). Example 18 showed that MpRNCIntermediate stimulated TLR2. MpRNC Intermediate also contains both RNAand DNA (Example 10). The ability of MpRNC Intermediate to stimulateTLR2, TLR3, TLR4, TLR5, TLR7, TLR8 and TLR9 was therefore determined tosee if the presence of RNA and DNA in MpRNC Intermediate resulted inactivation of nucleic acid-specific TLRs, or of any other TLR. It is tobe understood that MpRNC Intermediate is used as an example of MRNC, andthat the principles taught in this Example are applicable to MRNC fromthe mycobacteriaceae family.

The TLR stimulating activity of MpRNC Intermediate was tested on HEK293cells that are engineered to express one specific TLR (InvivoGen).Activation of a given TLR results in the NF-κB driven synthesis of SEAP,the enzymatic activity of which is detected in the supernatant asdescribed in Example 19. Treatment of HEK293 cells expressing thedifferent TLRs with MpRNC Intermediate was carried out as described inExample 18 using a final concentration of 90 μg/mL. The positive controlTLR agonists used in this study are shown in Table 20, as well as thefinal concentration used in the assay.

TABLE 20 TLR agonist positive controls TLR Agonist Final concentrationTLR2 Heat-killed  10⁸ cells/mL Listeria monocytogenes TLR3 Poly(I:C) 1.0μg/mL TLR4 Escherichia coli K12 LPS 0.1 μg/mL TLR5 Staphylococcustyphimurium 0.1 μg/mL flagellin TLR7 Gardiquimod 1.0 μg/mL(imidazoquinoline TLR7 agonist) TLR8 CLO75 (thiazoloquinolone 1.0 μg/mLTLR8 agonist) TLR9 CpG oligonucleotide 2006 0.1 μg/mL

The results of the evaluation are shown in Table 21. All of the TLRagonist controls gave positive responses. Significant stimulation ofHEK293 cells expressing TLR2 was seen with MpRNC Intermediate, andalthough some weak stimulation of HEK293 cells expressing TLR3 or TLR5was observed this was significantly lower than the respective positivecontrols, poly(I:C) and flagellin (3% and 4% of the positive controlactivity respectively). The apparent stimulation of TLR3 and TLR5 wassubsequently shown not to be reproducible or dose-related, and notactive at the concentrations of the standards, and was thereforeconsidered to be an artifact. MpRNC Intermediate did not stimulate anyother TLR, including RNA-specific TLR7 and TLR8, as well as the DNACpG-motif specific TLR9.

TABLE 21 TLR agonist activity of MpRNC Intermediate SEAP activity, OD630 HEK293 expressing TLR: MpRNC Intermediate* Positive control 2 3.7873.315 3 0.101 3.212 4 −0.001 2.205 5 0.129 3.231 7 0.042 2.384 8 0.0203.446 9 0.026 2.334 *Mean of 4 manufactured samples tested.

Three conclusions are to be drawn from these data. First, MpRNC onlyactivates the TLR TLR2; second the presence of RNA in MpRNC Intermediatedoes not result in activation of the RNA-specific TLRs TLR3, TLR7 orTLR8; and third that the presence of DNA in MpRNC Intermediate does notresult in activation of the CpG receptor TLR9, demonstrating an absenceof functional CpG motifs in MpRNC Intermediate.

Example 21 Impact of Intact Mycobacterium phlei Cells on the ImmuneStimulatory Activity of MpRNC Compositions

The impact of the presence of intact Mycobacterium phlei cells on theimmune stimulatory activity of MpRNC prepared as described in example 2was determined using a cytokine/chemokine-induction assay. Specifically,the immune stimulant activity of MpRNC compositions containing variousproportions of intact Mycobacterium phlei cells was determined. It is tobe realized that this example is used as an illustrative andrepresentative example of the influence of intact mycobacterial cells onthe immune stimulatory activity of MRNC. It is to be understood thatMpRNC is used as an example of MRNC, and that the principles taught inthis Example are applicable to MRNC from the mycobacteriaceae family.

Peripheral blood mononuclear cells (PBMC) were isolated from 6 healthyindividuals by density-centrifugation on Ficoll-Hypaque (GE Healthcare,Ste-Anne-de-Bellevue, Québec, Canada). Anticoagulated blood (lithiumsalt of heparin) was centrifuged at low speed to remove platelets, andPBMC were isolated by cushion centrifugation using Ficoll-Hypaque. Theisolated PBMC were washed by low-speed centrifugation (150×RCF for 10min at 4° C.) in RPMI 1640 (Wisent Inc., St Bruno, Québec, Canada)containing 10% heat-inactivated fetal bovine serum (Wisent Inc.) and 10μg/mL gentamycin (Sigma-Aldrich, Oakville, Ontario, Canada), andsuspended at a concentration of 5×10⁵ viable cells/mL medium. One mL wasplated in the wells of 24-well tissue culture plates. The cells wereallowed to incubate for 24 hours at 37° C. in an atmosphere of 5% CO₂.MpRNC Intermediate or MpRNC Intermediate containing varying amounts ofheat treated Mycobacterium phlei cells (121° C., 30 min) were added tothe tissue culture plates (final concentration 10 μg/mL for IL-10induction, 1 μg/mL for IL-12p40 induction), and the cells incubated at37° C. in an atmosphere of 5% CO₂ for a further 48 hours. Thesupernatants were removed, clarified by 0.2 micron membrane filtrationand the levels of IL-10 and IL-12p40 determined by kit ELISA (BioSource,Camarillo, Calif., USA, catalog numbers KHC0102 and KHC0122respectively) using the suppliers' recommended procedures.

The results are shown in FIG. 8. It can be seen that MpRNC Intermediate(containing a calculated 0.7% w/w intact mycobacteria), containingvarying proportions of intact autoclaved Mycobacterium phlei cells (therange tested being 100% MpRNC Intermediate through to 100% intactautoclaved Mycobacterium phlei cells to reflect the potential levels ofintact mycobacteria inherent in the new manufacturing procedures andcompositions) induced IL-10 production (FIG. 8 a) with a bell-shapedresponse, with the optimal proportion of intact autoclaved Mycobacteriumphlei cells being in the range of about 5 to 30% w/w. IL-10 inductionwith MpRNC Intermediate containing intact autoclaved Mycobacterium phleicells in the proportion of about 5-30% w/w was found to be higher thanthe 100% MpRNC Intermediate composition or a composition comprising 100%intact autoclaved Mycobacterium phlei cells.

FIG. 8 b shows that the MpRNC Intermediate composition containingvarying proportions of intact autoclaved Mycobacterium phlei cells(range tested: 100% MpRNC Intermediate through to 100% intact autoclavedMycobacterium phlei) induced IL-12p40 production with a bell-shapedresponse, with the optimal proportion of intact autoclaved Mycobacteriumphlei cells being in the range of about 10-50% w/w. IL-12 induction withMpRNC Intermediate composition containing intact autoclavedMycobacterium phlei cells in the proportion of about 10 to 50% w/w washigher than a composition comprising 100% MpRNC Intermediate or acomposition comprising 100% intact autoclaved Mycobacterium phlei cells.

Analysis of variance (ANOVA, two-way without replicates) showed that theproportion of intact Mycobacterium phlei cells in MpRNC significantlyinfluenced cytokine induction (p<0.025 at an alpha of 0.05), and thatthe levels of IL-10 and IL-12 induced by MpRNC were statisticallydifferent (p<0.05E⁻⁶ at an alpha of 0.05).

Example 22 MpRNC Induces Chemokine and Cytokine Synthesis in ImmuneEffector Cells

The chemokine- and cytokine-inducing activity of MpRNC was analyzed bythe use of a Milliplex® chemokine/cytokine analysis kit. It is to beunderstood that MpRNC is used as an example of MRNC, and that theprinciples taught in this Example are applicable to MRNC from themycobacteriaceae family.

Human PBMC were isolated by Ficoll density-gradient centrifugation ofwhole blood (13 healthy individuals in total). PBMC were prepared inRPMI 1640 medium containing 10% v/v heat-inactivated fetal bovine serum(56° C./30 min) (both from Wisent, St-Bruno, Québec, Canada) and 50μg/mL gentamicin sulfate (Sigma-Aldrich Canada, Oakville, Ontario,Canada). PBMC (1×10⁶ cells) were seeded in a volume of 1.0 mL in 6-wellflat-bottomed microplates and incubated at 37° C./5% CO₂/100% humidityin the cell culture medium described above with or without (untreatedcontrols) 160 μg/mL final concentration MpRNC Intermediate. Thecytokine, chemokine and hematopoietic growth factor levels in theculture medium were measured after 48 hours incubation using aMilliplex® MAP kit for human chemokines/cytokines (MilliporeCorporation, Billerica, Mass., USA) on a Bio-Plex 200 system (BioRadLaboratories, Hercules, Calif., USA). The results shown are expressed asthe mean fold-increase in chemokine/cytokine levels versus untreatedPBMC.

The results shown in Table 22 demonstrate that MpRNC Intermediate hasthe ability to stimulate a range of chemokines, cytokines and cellulargrowth factors from immune effector cells present in PBMC, thusdemonstrating its ability to act as an immune stimulant for theinduction of chemokines, cytokines and cellular growth factors.

TABLE 22 Chemokine, cytokine and cellular growth factor induction byMpRNC Intermediate Mean ± SD fold increase Chemokine/Cytokine Familyversus untreated PBMC G-CSF Hematopoietic growth 128.5 ± 101.4 factorGM-CSF Hematopoietic growth 41.0 ± 27.0 factor IL-1a (alpha) Cytokine16.2 ± 9.6  IL-1b (beta) Cytokine 116.3 ± 151.3 IL-10 Cytokine 23.4 ±12.5 TNF-a (alpha) Cytokine 42.4 ± 37.3 IL-6 Cytokine 254.6 ± 374.7MIP-1b (beta) Chemokine 39.2 ± 50.3 MIP-1a (alpha) Chemokine 27.3 ± 33.9GRO Chemokine 36.9 ± 61.1 MDC Chemokine 16.5 ± 26.4

Example 23 Stimulation of GM-CSF, MU-CSF, M-CSF, G-CSF, SDF-1a and LIFSynthesis in Human Peripheral Blood Mononuclear Cells by MpRNC

Multi-Colony Stimulating Factor (MU-CSF; also known as IL-3), whichstimulates the differentiation of pluripotent hematopoietic cells intomyeloid progenitor cells in addition to stimulating the production oferythrocytes, dendritic cells, granulocytes, megakaryocytes andmonocytes, Macrophage Colony Stimulating Factor (M-CSF),Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF),GranulocyteColony Stimulating Factor (G-CSF), Stromal Cell Derived Factor-1alpha(SDF-1a) and Leukemia Inhibitory Factor (LIF) are human hematopoieticgrowth factors showing a wide range of biologic activities that includethe growth promotion and cell differentiation of different types oftarget cells (progenitor cells), stimulation of hematopoiesis and stemcell mobilization. The ability of MpRNC Intermediate to stimulate thesynthesis of these growth factors from immune cell was determined. It isto be understood that MpRNC Intermediate is used as an example of MRNC,and that the principles taught in this Example are applicable to MRNCfrom the mycobacteriaceae family.

Peripheral blood mononuclear cells (PBMC) were isolated from 6 healthyindividuals by density-centrifugation on Ficoll-Hypaque. PBMC wereincubated at 0.5×10⁶ cells per mL in 6-well tissue culture plates with1.6-160 μg/mL MpRNC Intermediate (prepared as described herein, seeTables 2 and 3) for 48 hours. Levels of MU-CSF, M-CSF, GM-CSF, G-CSF,SDF-1a and LIF were measured after 48 hours in the supernatants using aBioplex® system (Bio-Rad, Mississauga, Ontario, Canada) with appropriatebeads and antibodies using Bi-Rad or Millipore reagents. An increase of50% in the level of the growth factor in the supernatant followingtreatment of the PBMC with MpRNC Intermediate versus control-treatedcells (≧1.5-fold increase) was used to define a positive stimulatoryeffect.

Table 23 shows that MpRNC Intermediate induced a dose-relatedstimulation (>1.5-fold increase) in the synthesis of all the growthfactors measured.

TABLE 23 Stimulation of GM-CSF, MU-CSF, M-CSF, G-CSF, SDF-1a and LIFsynthesis in human PBMC by MpRNC Intermediate Fold increase in growthfactor* Growth MpRNC Intermediate (μg/mL) factor 1.6 16 160 MU-CSF 2.6 ±3.6 3.7 ± 4.3 14.4 ± 7.3  M-CSF 2.0 ± 0.5 3.5 ± 1.2 5.0 ± 1.5 GM-CSF 1.0± 0.0 1.5 ± 1.5 24.1 ± 26.8 G-CSF 3.5 ± 3.9 20.9 ± 22.5 197.0 ± 163.6SDF-1a 2.6 ± 1.5 3.8 ± 2.5 3.8 ± 2.7 LIF 1.5 ± 0.7 1.8 ± 0.8 3.4 ± 3.4*The fold increase is expressed as the mean ± SD, n = 6

The data from Table 23 shows that MpRNC stimulates the synthesis ofhematopoietic growth factors from immune cells found in peripheralblood, with the greatest fold increases being seen with GM-CSF andG-CSF, followed by the pluripotent growth factor MU-CSF. M-CSF, SDF-1aand LIF also showed increases above the defined response thresholdlevel.

Example 24 Stimulation of GM-CSF, MU-CSF and LIF Synthesis in NormalHuman Urinary Tract Epithelial Cells by MpRNC

The ability of MpRNC Intermediate to stimulate growth factor synthesisby non-immune cells was examined using human urinary bladder epithelialcells. These were used as an example of epithelial cells in general. Itis to be understood that MpRNC Intermediate is used as an example ofMRNC, and that the principles taught in this Example are applicable toMRNC from the mycobacteriaceae family.

The normal human urinary tract epithelial cell line SV-HUC-1 (obtainedfrom the ATCC, Manassas, Va.) was incubated at 1.0×10⁵ cells per mLtissue culture medium (as recommended by the ATCC) in 96-well tissueculture plates with 0.016-160 μg/mL final concentration MpRNCIntermediate (prepared as described in Example 3) for 48 hours. MU-CSF,M-CSF, GM-CSF, G-CSF, SDF-1a and LIF were measured after 48 hours in thesupernatants using a Bioplex® system (Bio-Rad Laboratories (Canada)Inc., Mississauga, Ontario, Canada) with appropriate Milliplex® beadsand antibodies (Millipore Corporation, Billerica, Mass., USA). Anincrease of 50% in the level of growth factor versus control-treatedcells was used to identify positive stimulation following MpRNCIntermediate treatment 1.5-fold increase).

Table 24 shows the increase in growth factor production from normalhuman bladder epithelial cells following treatment with MpRNCIntermediate.

TABLE 24 Stimulation of growth factor synthesis in human SV-HUC-1 cellsfollowing treatment with MpRNC Intermediate Fold increase in growthfactor Growth MpRNC Intermediate (μg/mL) factor 0.016 1.6 160 MU-CSF 0.62.5 1.6 M-CSF 1.2 1.1 1.1 GM-CSF 1.0 1.0 4.0 G-CSF 1.0 1.0 1.0 SDF-1a1.0 1.1 1.0 LIF 1.2 3.1 2.7

The data from Table 24 shows that treatment of normal human urinarytract epithelial cells with MpRNC Intermediate resulted in adose-related stimulation of the synthesis of MU-CSF (bell-shapedresponse), GM-CSF and LIF (bell-shaped response), as defined by thethreshold value of >1.5-fold increase

Example 25 Stimulation of GM-CSF and MU-CSF Synthesis in Human BladderCancer Cells by MpRNC

The ability of MpRNC Intermediate to stimulate growth factor synthesisin non-immune cells was examined using human urinary bladder cancercells. These were used as an example of cancer cells in general. It isto be understood that MpRNC Intermediate is used as an example of MRNC,and that the principles taught in this Example are applicable to MRNCfrom the mycobacteriaceae family.

The human bladder cancer cell lines SV-HUC-2 and T24 (both obtained fromthe ATCC, Manassas, Va.) were incubated at 1.0×10⁵ cells per ml in96-well tissue culture plates (using the tissue culture mediumrecommended by the ATCC) with 0.016-160 μg/ml final concentration MpRNCIntermediate for 48 hours. Levels of MU-CSF, M-CSF, GM-CSF, G-CSF,SDF-1a and LIF were measured after 48 hours in the supernatants using aBioplex® 200 system with appropriate beads and antibodies using Bio-Radand/or Millipore reagents. An increase of 50% in the level of growthfactor versus control-treated cells (≧1.5-fold increase) was used toidentify positive stimulation following MpRNC Intermediate treatment.

Table 25 shows the increase in growth factor production from SV-HUC-2cells following treatment with MpRNC Intermediate, and Table 23 showsthe increase in growth factor production from T24 cells followingtreatment with MpRNC Intermediate.

TABLE 25 Stimulation of Growth Factor synthesis in human SV-HUC-2 cellsfollowing treatment with MpRNC Fold increase in growth factor GrowthMpRNC Intermediate (μg/mL) factor 0.016 1.6 160 MU-CSF 1.3 3.4 2.4 M-CSF1.0 1.0 1.1 GM-CSF 1.0 1.0 1.9 G-CSF 1.0 1.0 1.0 SDF-1a 1.1 1.1 1.2 LIF1.3 1.2 1.2

It is apparent from the data shown in Table 26 that incubation of thehuman bladder cancer cell line SV-HUC-2 with MpRNC Intermediate resultedin a dose-related stimulation of the synthesis of GM-CSF and MU-CSF.

TABLE 26 Stimulation of Growth Factor synthesis in human T24 cellsfollowing treatment with MpRNC Fold increase in growth factor GrowthMpRNC Intermediate (μg/mL) factors 0.016 1.6 160 MU-CSF 0.6 1.4 0.8M-CSF 0.9 1.0 1.0 GM-CSF 0.9 1.5 2.0 G-CSF 1.0 1.0 1.0 SDF-1a 0.9 1.11.2 LIF 0.7 1.4 1.2

The data shown in Table 26 demonstrate that treatment of the humanbladder cancer cell line T24 with MpRNC Intermediate stimulates theinduction of GM-CSF synthesis in a dose-related manner (50% increase at1.6 μg/mL MpRNC and 100% increase at 160 μg/mL MpRNC). MpRNC thereforestimulates hematopoietic growth factor synthesis from cancer cells.

Example 26 Stimulation of Colony Stimulating Factors by MpRNC FollowingIntraperitoneal (IP) Administration in Mice

This example serves to show that MpRNC Intermediate stimulates theproduction of growth factors in vivo. It is to be understood that MpRNCIntermediate is used as an example of MRNC, and that the principlestaught in this Example are applicable to MRNC from the mycobacteriaceaefamily.

Groups of 2 female C57BL/6 mice were treated with 1.0 mg/kg body weightMpRNC Intermediate (prepared at a concentration of 1 mg/mL in water forinjection as described in Example 3) via the IP route. Levels ofgranulocyte-monocyte colony stimulating factor (GM-CSF) and granulocytecolony stimulating factor (G-CSF) in the sera were determined using aBioplex® system with appropriate beads and antibodies (Bio-Rad and/orMillipore reagents) 1, 4, 7 and 24 hours after injection. An increase of50% in the level of growth factor versus control-treated mice 1.5-foldincrease threshold level) was used to identify positive growth factorstimulation following MpRNC treatment.

Table 27 shows the increase in GM-CSF and G-CSF levels in the sera ofthe treated mice at various times following the injection of MpRNCIntermediate.

TABLE 27 Stimulation of GM-CSF and G-CSF synthesis following IPadministration of MpRNC Intermediate to female C57BL/6 mice Hours postFold increase (mean ± SD)* injection GM-CSF G-CSF 1 7.6 ± 9.3 16.5 ± 3.34 56.2 ± 28.6 307.1 ± 97.8 7 0.7 ± 0.5  296.2 ± 287.8 24 2.5 ± 2.1  50.3± 34.1 *2 mice per treatment group.

The data from Table 27 shows that MpRNC Intermediate stimulates thesynthesis of GM-CSF and G-CSF in vivo, peaking at 4 hours post-treatmentfor GM-CSF and at 4-7 hours post-treatment for G-CSF. The results showedthat there was a mean maximal 56-fold increase in GM-CSF serum levels 4hours after MpRNC Intermediate injection when compared withcontrol-treated mice, and a mean maximal 300-fold increase in G-CSFserum levels 4-7 hours post injection when compared with control mice.These data demonstrate that MpRNC Intermediate has immune stimulantactivity (colony stimulating factor induction) in vivo followingsystemic administration.

Example 27 Anti-Cancer Activity of MpRNC and Impact of IntactMycobacterium phlei Cells

This example serves to show that MpRNC has anticancer activity. It is tobe understood that MpRNC Intermediate is used as an example of MRNC, andthat the principles taught in this example are applicable to MRNC fromthe mycobacteriaceae family. The impact of the presence of intactMycobacterium phlei cells on the anticancer activity of MpRNCIntermediate or MpRNC Low as prepared in Example 3 (that is, using amycobacterial RNC cell wall formulation) was determined using a cancercell inhibition of proliferation assay. It is to be understood that thisexample is used as an illustrative and representative example of theinfluence of intact mycobacterial cells on the anticancer activity ofMRNC in general.

RT4 human bladder cancer cells (ATCC, Manassas, Va., catalog # HTB-2™)were plated in DMEM culture medium supplemented with 10%heat-inactivated FBS (both from Wisent) and 10 μg/mL gentamycin(Sigma-Aldrich) at a concentration of 5×10⁵ cells/ml in 96-well tissueculture plates (50 μL volume), and allowed to adhere to the surface ofthe wells overnight at a temperature of 37° C. in an atmosphere of 5%CO₂. Autoclaved Mycobacterium phlei cells (121° C., 30 min),heat-treated MpRNC intermediate or low (121° C., 30 min) or combinationsof autoclaved Mycobacterium phlei cells mixed with autoclaved MpRNCintermediate or low in a volume of 50 μL tissue culture medium wereadded to the cells to give final concentrations of 0.01, 0.1, 1.0 and 10μg/mL MpRNC. The cells were then incubated for 48 hours at 37° C. in anatmosphere of 5% CO₂. Anti-proliferative activity against the RT4bladder cancer cell line was determined using an MTT((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)reduction assay. Briefly, 10 μL of an MTT solution (5 mg/mL in PBS) wasadded to each well after 48 hours incubation, and incubation continuedfor a further 4 hours. The reaction was then stopped by the addition of100 μL isopropanol:HCl (24:1 v/v). The reduction of MTT leads to theproduction of formazan. Formazan was solubilized by use of amicropipette, and the optical density (O.D.) determined in a microplatereader at a wavelength of 570 nm against appropriate controls. Thepercentage inhibition of cell proliferation was determined using theequation:

% Inhibition=100−[(Test O.D.−Control O.D.)/Control O.D.].

The results obtained with MpRNC Low (see Example 3, Table 6; the thirdcomposition) plus controlled Mycobacterium phlei cell content and MpRNCIntermediate (Example 3, Table 6; the second composition) pluscontrolled Mycobacterium phlei cell content using the human bladdercancer cell line RT4 are shown in Tables 28 and 29 respectively. Potencydeterminations relative to Mycobacterium phlei were determined usingPharmPC v4.2 (Microcomputer Specialists, Philadelphia, Pa., USA).

TABLE 28 Inhibition of RT4 bladder cancer cell proliferation byautoclaved MpRNC low, autoclaved Mycobacterium phlei or a combination ofautoclaved MpRNC (low) and autoclaved Mycobacterium phlei in variousproportions Mycobacterium % Inhibition of proliferation phlei:MpRNC Low:Concentration, μg/mL Relative (proportion) 0.01 0.1 1.0 10 Potency100:0  17.6 18.1 25.8 33.8 1 99:1  11.9 16.2 24.2 34.5 2.5 90:10 18.320.0 30.4 35.5 2.2 70:30 18.0 25.5 35.8 43.7 1 50:50 19.1 25.4 35.6 40.224.1 30:70 16.2 23.2 34.6 36.6 8.1 10:90 23.9 32.5 42.4 48.7 5.9  5:9520.6 30.6 43.01 46.7 37.4  1:99 20.2 29.7 41.9 46.3 145.  0:100 17.630.8 38.8 42.9 91.0

TABLE 29 Inhibition of RT-4 bladder cancer cell proliferation by MpRNCIntermediate, autoclaved Mycobacterium phlei or a combination ofautoclaved MpRNC Intermediate and autoclaved Mycobacterium phlei invarious proportions Mycobacterium % Inhibition of proliferationphlei:MpRNC Intermediate Concentration, μg/mL Relative (proportion) 0.010.1 1.0 10 Potency 100:0  8.2 16.9 24.7 33.3 1 99:1  13.2 17.8 27.6 37.32.6 90:10 15.8 22.0 34.4 40.4 11.6 70:30 20.6 24.2 37.5 38.8 6.2 50:5022.3 27.5 38.6 43.4 72.0 30:70 18.9 26.0 33.1 32.4 8.8 10:90 22.6 33.238.8 43.2 37.1  5:95 19.4 32.5 40.9 43.5 39.7  1:99 23.6 34.6 43.1 47.9151.0  0:100 24.4 33.0 43.0 47.1 263.3

The results show that MpRNC Low and MpRNC Intermediate have anticanceractivity, and that autoclaved Mycobacterium phlei (i.e., a preparationthat comprises intact mycobacterial cells) has less anti-proliferativeactivity against RT4 bladder cancer cells than MpRNC containing eitheradded Mycobacterium phlei equivalent to that in MpRNC low or MpRNC high.Moreover, MpRNC compositions with intact Mycobacterium phlei had lessthan optimum anti-proliferative activity. Calculation of the relativepotency of MpRNC without added intact mycobacterial cells relative toMpRNC with increasing intact mycobacterial cell content demonstratedthat both mycobacterial RNCs were considerably more potent than intactMycobacterium phlei (91-fold and 263-fold for MpRNC Low and Intermediaterespectively). The addition of intact mycobacterial cells resulted in asignificant decrease in potency when 10% w/w or greater, and the optimalintact mycobacterial cell content was <1.0% w/w depending on the initialdegree of intact mycobacterial cell content (0% for MpRNC Intermediate,and 1% for MpRNC Low. MRNC compositions and formulations intended foranticancer applications therefore benefit from a low intactmycobacterial cell content.

Example 28 Anticancer Activity of MpRNC Intermediate Towards Lewis LungCarcinoma

This example serves to show that MpRNC Intermediate possesses anticanceractivity both in vitro and in vivo. It is to be understood that MpRNCIntermediate is used as an example of MRNC, and that the principlestaught in this Example are applicable to MRNC from the mycobacteriaceaefamily.

The direct anticancer activity of MpRNC Intermediate manufacturedaccording to the method of the present invention and formulated inRNase-free water was determined using the Lewis lung carcinoma (LLC)cell line. LLC cells were obtained from the ATCC (Manassas, Va., USA).LLC is used extensively in the development of chemotherapeutic andimmunotherapeutic anticancer agents (see for example Kimura, Y. NewAnticancer agents: In vitro and in vivo evaluation of antitumor andantimetastatic action of various compounds isolated from medicinalplants. in vivo 2005; 19:37-60). The LLC cells were maintained in DMEMmedium containing 10% v/v heat-inactivated fetal bovine serum (56° C.for 30 min) (both from Wisent, St-Bruno, Québec, Canada), and 10.0 μg/mLgentamycin sulfate (Sigma-Aldrich, Oakville, Ontario, Canada) at 37° C.in an atmosphere of 5% CO₂. LLC cells in DMEM/10% heat-inactivated FBSwithout gentamycin were seeded at 4.0×10⁴ cells in a volume of 0.1 mL in96-well flat-bottomed tissue culture microplates, and MpRNC Intermediatesuspension (0.1 mL) was added to triplicate wells to give finalconcentrations of 0.01-100.0 μg/mL. The plates were then incubated for48 hours at 37° C. in an atmosphere of 5% CO₂. The antiproliferativeactivity of MpRNC was determined as described for the RT4 bladder cancercell line (formazan reduction) in Example 27. The results are shown inTable 30.

TABLE 30 Inhibition of LLC cellular proliferation by MpRNC IntermediateMpRNC Intermediate (μg/mL) 0.01 0.1 1.0 10 100 % inhibi- 9.5 ± 4.9 23.0± 5.7 38.5 ± 9.2 45.0 ± 11.3 49.0 ± 5.7 tion of LLC pro- liferation Theresults shown are the mean ± SD of 2 experiments.

The results shown in Table 30 demonstrate that MpRNC has dose-relatedanticancer activity against LLC cells, as determined by measurement ofinhibition of proliferation. Example 27 and this Example (Example 28)therefore demonstrate that the direct anticancer activity of MpRNC isnot cancer cell type- and by extension not cancer specific.

Distant site metastases are the leading cause of cancer-associatedmortality (Kim et al., Carcinoma produced factors activate myeloid cellsvia TLT2 to stimulate metastasis. Nature 2009; 457:102-106).Experimental liver metastases can be produced by the injection of LLCcells into the spleen (Ligo et al., Therapeutic activity and tissuedistribution of ME2303, a new anthracycline containing fluorin, and itsmetabolites in mice bearing hepatic metastases of Lewis lung carcinoma.Anti-Cancer Drugs 1990; 1:77-82 and Alino et al Morpho-functional studyof vascular fluorochrome delivery to lung and liver metastases of Lewislung carcinoma (3LL), Tumori 1991; 77:206-211). The tumor loci inducedin the liver by the intrasplenic injection of LLC cells are regarded asa model of metastatic spread to the liver, and where the number of tumorfoci is increased by blocking the activity of Kupffer cells (specializedmacrophages lining the sinusoids of the liver) with carrageenan. Thismodel therefore provides a useful tool for studying different aspects ofliver metastases, (Kopper et al., Experimental model for livermetastasis formation using Lewis lung tumor. J. Cancer Res and Clin.Oncol. 1982; 103:31-38). The objective of the following studies was todetermine the efficacy of MpRNC suspension, following intravenous (IV)administration, as a treatment for experimental hepatic metastasesinduced by the intrasplenic injection of LLC cells in C57BL/6 mice.

LLC cells were maintained in DMEM medium containing 10% v/vheat-inactivated fetal bovine serum as described above. The cells wereserially passaged every 3-4 days, and were used at passage 2-3. Thecells were harvested, by trypsinization (4 min at 37° C.), washed twicein DMEM medium without fetal bovine serum and gentamycin sulfate,centrifuged at 250×g for 5 min at 4° C. and the cell pellet suspended inDMEM medium without fetal bovine serum and gentamycin sulfate. Thenumber of cells was adjusted to the required concentration after cellviability was determined using trypan blue exclusion and counting in ahemacytometer. The cells were kept on ice until intrasplenic injection.

Female C57BL/6 mice, 10-12 weeks of age, were obtained from CharlesRiver Laboratories Ltd., St. Constant, Québec, Canada. This mouse strainis syngeneic for LLC cells. Hepatic metastases were induced by theintrasplenic injection, under general anesthesia, of LLC cells(1.0-3.0×10⁵ LLC cells in a volume of 100 μL). The spleen was excisedafter cancer cell injection. The effect of treating the mice prior toLLC injection (prophylactic treatment) or after LLC cell injection(therapeutic treatment) was firstly examined. MpRNC Intermediate wasinjected 1 day before (prophylactic regimen), on days 2, 5, 7, 9 and 12(therapeutic regimen), or on days −1, 2, 45, 7, 9 and 12 (prophylacticand therapeutic regimen) following LLC cell injection using groups of 10mice. MpRNC Intermediate formulated in RNase-free water for injection asdescribed in Example 3 was administered in a dose volume of 100 μL,corresponding to a dose of 5 mg/kg body weight. The number of hepaticmetastases was determined on day 15 following LLC cell injection. Theresults (Table 31) show that the therapeutic+prophylactic administrationprotocol was the most effective treatment regimen, followed by theprophylactic treatment regimen, and that the therapeutic treatmentregimen was the least effective. Thus a combination of prophylactic andtherapeutic therapy is the most effective for the treatment of hepaticLLC metastases.

TABLE 31 Treatment of experimental LLC hepatic metastases by MpRNCIntermediate Group 4: Group 2: Group 3: MpRNC MpRNC MpRNC IntermediateGroup 1: Intermediate Intermediate Prophylactic + Untreated ProphylacticTherapeutic Therapeutic Mean number 26 11 21 6 of tumors

These data demonstrate that the use of MRNC Intermediate as a standalone therapy or in neoadjuvant and adjuvant settings for the treatmentof cancer would be beneficial in reducing tumors resulting from themetastasis of primary carcinoma. The results shown in Table 31 areconsistent with activation of host-defense mechanisms in the targetorgan prior to exposure to cancer cells (prophylactic activity), thusresulting in decreased tumor cell seeding in the liver, as well as atherapeutic effect on the cancer cells following their initialimplantation in the target organ (therapeutic activity). Thedemonstration of direct anticancer towards LLC cell targets demonstratedin Table 30 supports such a mode of action.

A second study was conducted to determine the effect of MpRNCIntermediate suspension treatment on LLC hepatic metastases using theprophylactic/therapeutic regimen in a larger number of animals.Experimental LLC hepatic metastases were established as described aboveusing groups of 20 mice, and prophylactic/therapeutic treatment withMpRNC Intermediate at a dose of 5 mg/kg body weight carried out.

The results of this study showed that there were 141±72 (mean±SD)metastases in the control group, and that by using a prophylactic andtherapeutic treatment protocol as described above, MpRNC Intermediatetreatment significantly reduced the number of metastases to 62±41(mean±SD, p<0.0002, Mann-Whitney U-test), thus demonstrating asignificant anticancer activity in vivo against experimentally-inducedhepatic metastases. The anticancer activity demonstrated against LLCcells in vitro is therefore confirmed as a predictive measure of theinhibitory effect against the development of hepatic metastases in vivo.

Example 29 Anti-Cancer Activity of Mycobacterial RNC

This example serves to show that MRNC possess anticancer activity. It isto be understood that the principles taught in this Example areapplicable to MRNC from the mycobacteriaceae family.

The anticancer activity of the mycobacterial RNC as prepared in Example3 and 4 was determined using an inhibition of cancer cell proliferationassay. Those of skill in the art recognize that such studies are knownto be predictive of anticancer activity in vivo. In this example,bladder cancer cell lines were used as being representative of cancercell lines in general, and it is to be understood that one skilled inthe art will immediately appreciate that anticancer activity studiesusing such cancer cell lines can be readily extrapolated to other cancercell lines.

A total of four human bladder cancer cells were used in the example, andall were obtained from the ATCC (Manassas, Va., USA). Cultivation of thecell lines was carried out as recommended by the ATCC. Details of thebladder cancer cell lines are shown in Table 32.

TABLE 32 Bladder cancer cell line characteristics ATCC/E CACC Ethnicity/Molecular Cell Line NO. Sex/Age characteristics Reference HT-1376CRL-1472 Caucasian/ Mutated p53 & Rasheed et al. Transitional, F/58 yrsp21, lack of 1977. JNCI Grade-III pRb expression 58: 881. RT4 HTB-2Caucasian/ WT p53 & p21 Rigby & Franks. Papilloma, M/63 yrs 1970. Br. J.Benign Cancer 24: 746. SCaBER HTB-3 African/ No expression O'Toole etal. Squamous, M/58 yrs of pRb, 1976. Int. J. Grade mutated p53 Cancer.17: 707. unknown SW780 CRL-2169 Caucasian/ N/A Kyriazis et al.Transitional, F/80 yrs 1986. Cancer Grade-I Res. 44: 3997.

Bladder cancer line cells were plated in DMEM culture mediumsupplemented with 10% heat-inactivated FBS (both from Wisent) and 10μg/mL gentamycin (Sigma-Aldrich) at a concentration of 5×10⁵ cells/ml in96-well tissue culture plates (50 μL volume), and allowed to adhere tothe surface of the wells overnight at a temperature of 37° C. in anatmosphere of 5% CO₂. Mycobacterial RNC Intermediate prepared asdescribed in Example 3 and Example 4 in a volume of 50 μL tissue culturemedium was added to the cells to give final concentrations of 0.0016,0.016, 0.16, 1.6, 16, and 80 μg/mL MpRNC. The cells were then incubatedfor 48 hours at 37° C. in an atmosphere of 5% CO₂. Anti-proliferativeactivity was determined using an MTT((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)reduction assay. Briefly, 10 μL of an MTT solution (5 mg/mL in PBS) wasadded to each well after 48 hours incubation, and incubation continuedfor a further 4 hours. The reaction was then stopped by the addition of100 μL isopropanol:HCl (24:1 v/v). The reduction of MTT leads to theproduction of formazan. Formazan was solubilized by use of amicropipette, and the optical density (O.D.) determined using ELx-808IUmicroplate reader (BioTek Instrument, Winooski, Vt., USA) using the KCJunior software package (BioTek Instruments) at a wavelength of 570 nmagainst appropriate controls. The percent inhibition of cancer cellproliferation was determined using the equation:

% Inhibition=100−[(Test O.D.−Control O.D.)/Control O.D.].

The potency of the MRNC compositions was determined using PharmPC v4.2(Computer Associates, Philadelphia, Pa.)

The results obtained with the MRNC using the four human bladder cancercell lines are shown in Table 33. All MRNC compositions demonstrateddose-dependent anti-proliferative activity against the bladder cancercell lines. MbRNC from Mycobacterium bovis strain BCG was the leasteffective, demonstrating dose-related anti-proliferative activity, with˜20-40% inhibition (depending on the bladder cancer cell line) at thehighest dose tested. All of the other MRNC demonstrated dose-relatedanti-proliferative activity, with ˜35-70% inhibition (depending on thebladder cancer cell line) at the highest dose tested. For this reasonthe potency of the different MRNC relative to MRNC from Mycobacteriumbovis strain BCG was determined, and the results are shown in Table 33.

TABLE 33 Antiproliferative potency of MRNC relative to Mycobacteriumbovis BCG MbRNC Potency relative to Mycobacterium bovis BCG MbRNC Meanpotency, 4 Mycobacterial cancer cell lines RNC HT1376 RT4 ScaBER SW780(±SD) MbRNC 1 1 1 1 1 Intermediate MpRNC 716.00 1430.00 7590 6220 3987 ±3428 Intermediate MsRNC 214.00 145.00 637 1530 631 ± 635 IntermediateMvRNC 4190.00 1170.00 11900 9890 6794 ± 4978 Intermediate

The most potent MRNC for inhibition of bladder cancer proliferation wasthat obtained from Mycobacterium vaccae, followed in decreasing order ofpotency by MpRNC from Mycobacterium phlei, MsRNC from Mycobacteriumsmegmatis, and MbRNC from Mycobacterium bovis strain BCG. The magnitudeof and the order of potency for the mycobacterial RNC compositions wasmaintained for all 4 bladder cancer cell lines, irrespective of theirorigin (low-grade, high-grade) or mutational status, with Mycobacteriumvaccae MvRNC and Mycobacterium phlei MpRNC being some ˜6800-fold and˜4000-fold more potent respectively than Mycobacterium bovis BCG MbRNC.

It is to be realized that this example teaches three importantprinciples. Firstly, MRNC prepared from disparate mycobacterial species(pathogenic, non-pathogenic, fast-growing, slow growing) possessanticancer activity, and thus such activity is therefore applicable toMRNC prepared from member species of the mycobacteriaceae family.Secondly, the mutational status of the cancer cell target does notappear to play a role in determining the anticancer activity. In fact,significant anticancer activity is seen with cancer cell linespossessing known treatment resistant genotypes (p53 and p21 mutations)and MDR phenotypes (for example HT1376). Those skilled in the art knowthat such mutations and resistance phenotypes are characteristic ofcancer in general, and are not restricted to bladder cancer cells, andwill appreciate that the anticancer activity of MRNC is thereforeapplicable to many other cancer types. Thirdly, MRNC prepared from threefast-growing mycobacterial species is more effective than MRNC preparedfrom a slow growing mycobacterial species such as Mycobacterium bovisstrain BCG.

Example 30 Preparation of Mycobacterium avium SubspeciesParatuberculosis RNA-Containing Composition (MapRNC) and Determinationof Anticancer Activity

In this example, intact Mycobacterium avium subspecies paratuberculosiscells are first washed by low-speed centrifugation to remove culturemedium components, and are then disrupted using high-pressurehomogenization with an Avestin EmulsiFlex-05 high-pressure homogenizeras described in Example 3. After high-pressure homogenization theremaining intact mycobacteria are removed by differential centrifugationusing relative centrifugal forces that are optimized for the controlledremoval of any residual, intact and undisrupted mycobacterium. Themycobacterial cell depleted fraction, comprising nucleic acids of themycobacterium associated with mycobacterial cell wall fragments and thedesired and controlled amounts of intact mycobacterial cells for optimalanticancer activity, is further purified by centrifugation washing at ahigher relative centrifugal force, to remove soluble contaminants. TheMapRNC is isolated as a pellet following centrifugation washing at highrelative centrifugal force. The MapRNC is then heat-treated at 121° C.for between 5-30 min. The anticancer activity of MapRNC is determinedusing cancer cell lines representative of the major cancer cell types asdescribed in Examples 8 and 9. The results show that heat-treated MapRNChas dose-related anti-proliferative activity against cancer cell linesand against hepatic metastases.

Example 31 Anti-Cancer Activity of Total RNA of Mycobacterium phlei

The biological activity of a total RNA preparation from Mycobacteriumphlei cells was determined using a cancer cell proliferation assay. TheRNA was prepared using the Trizol reagent (Invitrogen, Carlsbad, Calif.,USA) according to the manufacturer's instruction following cell lysis inFastRNA Blue-Tubes (Bio-101 Inc.) using a FastPrep FP120 bead-beaterapparatus (Savant, Thermo-Fisher, Nepean, ON, Canada) for 1 min at level4.5. The RNA preparation was then treated with ‘DNA-free’ reagent toremove residual genomic DNA according to the manufacturer's instruction(Ambion, Austin, Tex., USA). RT4 (ATCC catalog # HTB-2) and HT1376 humanbladder cancer cells (ATCC catalog # CRL-1472) were plated in DMEMculture medium supplemented with 10% FBS (both from Wisent) and 10 μg/mlgentamycin (Sigma-Aldrich) at a concentration of 2×10⁵ cells/ml in96-well tissue culture plates (50 μL volume), and allowed to adhere tothe surface of the wells overnight at a temperature of 37° C. in anatmosphere of 5% CO₂. Total RNA in a volume of 50 μL tissue culturemedium was added to the cells to give final concentrations of 0.01, 0.1,1.0 μg/mL of RNA. The cells were then incubated for 72 hours at 37° C.in an atmosphere of 5% CO₂. Anti-proliferative activity against thecancer cell lines was determined using an MTT((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)reduction assay. Briefly, 10 μL of an MTT solution (5 mg/mL in PBS) wasadded to each well after 72 hours incubation, and incubation continuedfor a further 4 hours. The reaction was then stopped by the addition of100 μL isopropanol:HCl (24:1 v/v). The reduction of MTT leads to theproduction of formazan. Formazan was solubilized by use of amicropipette, and the optical density (O.D.) determined in a microplatereader at a wavelength of 570 nm against appropriate controls. Thepercentage inhibition of cell proliferation was determined using theequation:

% Inhibition=100−[(Test O.D.−Control O.D.)/Control O.D.].

The results show a moderate anti-cancer activity of total RNA ofMycobacterium phlei against HT1376 with inhibitory activity of 2.2%,7.1% and 6.8% at 0.1 μg/mL, 1.0 μg/mL and 10.0 μg/mL RNA respectively,and slightly more inhibitory activity against RT4 with an inhibition ofproliferation of 3.1%, 7.6% and 18.6% at 0.1 μg/mL, 1.0 μg/mL and 10.0μg/mL RNA respectively. When compared to the results obtained in Example27 or Example 29, where the mycobacterial RNA was in the form ofoligoribonucleotides and polyribonucleotides formulated with amycobacterial cell wall, it is clear that there are distinct andunexpected advantages to be obtained with respect to biological activitywhen using a mycobacterial cell wall RNA composition (or otherpharmaceutically acceptable carriers such as chitosan or cationicliposomes).

Example 32 Determination of the Integrated Potency Index ofMycobacterial Cell Wall RNC

Mycobacterial cell wall RNCs (MRNCs) of the present invention have theability to stimulate the immune system through 2 distinct receptorsystems and to inhibit cancer cell division, and are thus trifunctionaltherapeutic compositions. Using the data from Examples 29, 17 and 19(anti-proliferative potency, NOD2 activation potency and TLR2 activationpotency respectively), an Integrated Potency Index for MpRNCIntermediate, MsRNC Intermediate and MvRNC Intermediate relative toMbRNC Intermediate was determined as a means of integrating the overallanticancer and immune stimulant activities. Because of the wide range ofnumerical values for the relative potencies between the different assays(≧3 magnitudes), a natural logarithm (log) transformation was used inthis determination, and the values for each activity summed. MbRNC wasassigned a potency of 1, and the potency of the other MRNC determinedrelative to MbRNC. It is to be understood that the natural log of 1 is0. The results are shown in Table 34.

TABLE 34 Integrated Potency Index of MRNC Intermediate. Anti- Integratedproliferative NOD2 TLR2 Potency MRNC potency potency potency IndexIntermediate (natural log) (natural log) (natural log) (natural log)MbRNC 0 0 0 0 MpRNC 8.29 4.25 −0.45 12.09 MsRNC 6.45 −0.09 0.67 7.30MvRNC 8.82 0.37 1.32 10.52

It is clear that while all MRNC had similar, comparable potencies withrespect to TLR2 activation, MpRNC, MsRNC and MvRNC were all more potentthan MbRNC with respect to anticancer activity. MpNAC however was themost potent MRNC with respect to NOD2 activation. Determination of theIntegrated Potency Index showed that MpRNC has the highest overallpotency, followed by, in descending order, MvRNC, MsRNC and MbRNC.

Example 33 Preparation of MpRNC Compositions with Controlled Amounts ofIntact Mycobacterial Cells for Immune Stimulation

MpRNC is conveniently prepared with controlled amounts of intactautoclaved mycobacteria by preparing MpRNC Low (as detailed in Example 2and Example 3) and adding intact autoclaved mycobacterial cells to MpRNCto give the desired proportion optimal for the induction of immuneresponses. Generally, the inventors have found that the optimalproportion of intact Mycobacterium phlei cells needed to induce animmune response is within the range of about 5-50% by weight, with suchproportions giving rise to optimal immune stimulatory activity.

Example 34 Preparation of MRNC Compositions with Controlled Amounts ofIntact Mycobacterial Cells for Immune Stimulation

MRNC is conveniently prepared from a given mycobacterial species of themycobacteriaceae family such as but limited to Mycobacterium bovisstrain BCG, Mycobacterium avium substrain paratuberculosis,Mycobacterium smegmatis or Mycobacterium vaccae, by preparing MRNC Low(as described in Example 3 for MpRNC) and adding intact autoclavedmycobacterial cells to MRNC to give the desired proportion optimal forthe induction of immune responses. Generally, the inventors have foundthat the optimal proportion of intact mycobacterial cells needed toinduce an immune response is within the range of about 5-50% by weight,with such proportions giving rise to optimal immune stimulatoryactivity.

Example 35 Treatment of MpRNC with Ribonuclease-A

Treatment of MpRNC with ribonuclease (for example, the enzymaticdigestion conditions described in Example 10) results in a significantreduction in the amount of RNA contained within an MRNC or MpRNCcomposition. Such reductions significantly reduce the ability of MRNC orMpRNC to act as therapeutic agents.

MpRNC Intermediate was made as described in Example 3 except that beforethe final two cycles of high-pressure homogenization, the washed MpRNCIntermediate pellet was resuspended in water for injection with orwithout RNase A (Sigma, final concentration 10 μg/mL; MpRNC-RNase andMpRNC-Control respectively) for 3 hours at 37° C. The MpRNCs were thenwashed by high-speed centrifugation to remove RNase A and resuspended inwater for injection at a concentration of 1 mg/mL. The suspensions weresubsequently homogenized and terminally sterilized. The anticanceractivity of MpRNC-RNase and MpRNC Control was determined using thebladder cancer cell lines HT1376 and RT-4 as described in Example 27.The relative potency of the two MpRNC formulations was calculated fromthe linear part of the dose-response curve using PharmPC v4.2(Microcomputer Specialists, Philadelphia, Pa., USA).

Both MpRNC formulations inhibited the proliferation of HT1376 and RT-4bladder cancer cells in a dose-related manner (Table 35 and 36respectively). RNase treatment however resulted in a reduction in theanti-proliferative activity of MpRNC towards both bladder cancer celllines.

TABLE 35 RNase treatment reduces the anti-proliferative activity ofMpRNC towards HT1376 bladder cancer cells MpRNC concentration %Inhibition (μg/mL) MpRNC-Control MpRNC-RNase treated 0 0.00 0.00 0.001614.29 8.51 0.016 15.51 10.97 0.16 25.99 16.31 1.6 45.71 37.52 16 56.6050.65

TABLE 36 RNase treatment reduces the anti-proliferative activity ofMpRNC towards RT-4 bladder cancer cells MpRNC concentration % Inhibition(μg/mL) MpRNC-Control MpRNC-RNase treated 0 0.00 0.00 0.0016 11.05 1.470.016 16.94 7.83 0.16 27.90 14.87 1.6 43.39 36.20 16 49.82 42.86

The anti-proliferative potency of MpRNC Intermediate-RNase treatedrelative to MpRNC Intermediate-Control treated against HT-1376 and RT-4bladder cancer cell lines is shown in Table 37.

TABLE 37 RNase treatment reduces the anticancer potency of MpRNCIntermediate Bladder cancer Potency of RNase-treated cell line MpRNCrelative to MpRNC HT1376 0.25 RT-4 0.14

Removal of RNA in MpRNC Intermediate as a result of RNase treatmentresulted in a considerable reduction in anticancer potency (4-fold and7-fold for HT1376 and RT-4 bladder cancer cells respectively), thusdemonstrating that preservation of the RNA in MpRNC is a requirement foroptimal anticancer activity.

Example 36 Formulation of BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC with Cationic Liposomes and Determination of Anticancer Activity

All of the following procedures are conducted under sterile asepticconditions. BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC areprepared by disruption of intact bacteria, mycobacteria or M. phleiusing high-pressure homogenization, removal of undisrupted bacteria,mycobacteria or M. phlei by centrifugation at low RCF, and extraction ofthe RNC composition using phenol/chloroform/isoamyl alcohol and ethanolprecipitation. The extracted RNA in the RNC composition are reduced inchain length by heat treatment at 121° C. for 5 min to prepareoligoribonucleotides and polyribonucleotides containing betweenapproximately 20-40 bases. Cationic liposomes composed of1,2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2-dioleoyl-3-trimethylammonium-propane(DOPE/DOTAP) in the molar ratio 1:1 are prepared by dissolving thephospholipid and cationic lipid in anhydrous chloroform (1 mg/mL)followed by rotary evaporation under reduced pressure in round-bottomedflasks to form a thin phospholipid/lipid film. Control liposomes areprepared by adding the required volume of NaCl (0.85% w/v) to the thinphospholipid/lipid film, and agitating at 65° C. to form liposomes.Cationic liposomes containing BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNCand MvRNC are prepared by adding a NaCl solution containing BRNC, MRNC,MpRNC, MbRNC, MsRNC, MapRNC and MvRNC at a concentration of 0.1 mg/mL tothe thin phospholipid/lipid film, and agitating at 65° C. to formliposomes. The cationic liposomes containing BRNC, MRNC, MpRNC, MbRNC,MsRNC, MapRNC and MvRNC will have a mean diameter of approximately 825nm and a ζ-potential of approximately +36 mV. The murine B16 melanomainhibition of proliferation assay is carried-out as described in example27, using a liposomal BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNCconcentration range of 0.01 to 10 μg/mL. Control incubations areconducted using the corresponding concentrations of control cationicliposomes or of non-liposomal BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNCand MvRNC. MTT reduction is carried-out as described in example 27, andthe amount of inhibition of cell proliferation determined. The resultsshow that cationic liposomes containing BRNC, MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC inhibit the proliferation of B16 melanoma cells in adose-dependent manner, and that the cationic liposomes alone have noinhibitory activity. The inhibitory activity of the cationic liposomescontaining BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC issignificantly greater than BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC alone. These data demonstrate that a cationic liposome formulationacts as a pharmaceutical delivery system for BRNC, MRNC, MpRNC, MbRNC,MsRNC, MapRNC and MvRNC.

It is to be understood that RNA may be isolated from gram-negative andgram-positive bacteria, and from mycobacteria, Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis and Mycobacterium vaccae, and formulatedwith the cationic liposomes as described in this example without addingthe cell walls from these species.

It is to be realized that B16 melanoma cells are used in this example asbeing representative of a typical cancer cell, and that cationicliposomes are used in this example as a as a typical pharmaceuticaldelivery system for NA, and that one skilled in the art will appreciatethe applicability using such types of pharmaceutical delivery systemsand formulations in the treatment of cancer in general.

Example 37 Formulation of BRNC, MRNC, MRNC, MbRNC, MsRNC, MapRNC andMvRNC with Chitosan Nanoparticles and Determination of AnticancerActivity

All of the following procedures are conducted under sterile asepticconditions. BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC areprepared by disrupting of intact bacteria, mycobacteria or M. phleiusing high-pressure homogenization, removing undisrupted bacteria,mycobacteria or M. phlei by centrifugation at low RCF, and extractingthe RNA using phenol/chloroform/isoamyl alcohol and ethanolprecipitation. The extracted RNA is reduced in chain length byautoclaving at 121° C. for 5 min to prepare oligoribonucleotides andpolyribonucleotides containing between approximately 20-40 bases.Chitosan nanoparticles are prepared by mixing equal volumes oftripolyphosphate (0.5 mg/mL in water) and chitosan (average molecularweight 500,000, concentration 1 mg/mL in water) for 2 min at 20° C. toform control chitosan nanoparticles or by mixing equal volumes ofbacterial, mycobacterial or M. phlei oligoribonucleotides andpolyribonucleotides (BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC,0.5 mg/mL in water) and chitosan (average molecular weight 500,000,concentration 1 mg/mL in water) for 2 min at 20° C. The incorporation ofBRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC in the chitosannanoparticles is 100%. The chitosan nanoparticles are washed bycentrifugation and their size and ζ-potential (an indirect measure ofthe particle surface charge) determined. The chitosan nanoparticles havea size in the range of approximately 200 to 1000 nm and a ζ-potential ofapproximately −20 to −35 mV. B16 melanoma cells are grown to confluencein MEM supplemented with MEM none-essential amino acids, gentamycin (50μg/mL) and 10% v/v heat-inactivated fetal serum at 37° C. and 5% CO₂.The cells are harvested by trypsinization, resuspended in MEM tissueculture medium and plated in the wells of tissue culture plates (96 wellplates, 100 μL containing 5×10³ cells) and allowed to adhere for 3 hoursat 37° C. in 5% CO₂. Chitosan nanoparticles containing BRNC, MRNC,MpRNC, MbRNC, MsRNC, MapRNC and MvRNC are then added to the B16 melanomacells (ATCC) to give a final concentration of BRNC, MRNC, MpRNC, MbRNC,MsRNC, MapRNC and MvRNC in the range 0.001 to 100 μg/mL. Controlchitosan nanoparticles or BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC without chitosan nanoparticles are added to the B16 melanoma cellsto give concentrations comparable to the BRNC, MRNC, MpRNC, MbRNC,MsRNC, MapRNC and MvRNC chitosan nanoparticles. The murine B16 melanomacells are incubated for 48 hours at 37° C./5% CO₂, and the cell growthover this time determined using an MTT reduction assay. The level ofreduced MTT, which corresponds to the number of viable cells, isdetermined at 570 nm using an ELISA plate reader. The results show thatchitosan nanoparticles containing BRNC, MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC inhibit the proliferation of B16 melanoma cells in adose-dependent manner, that the chitosan nanoparticles alone have noinhibitory activity, and that the inhibitory activity of the chitosannanoparticles containing BRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC andMvRNC is significantly greater than BRNC, MRNC, MpRNC, MbRNC, MsRNC,MapRNC and MvRNC alone. These data demonstrate that a chitosannanoparticle formulation acts as a pharmaceutical delivery system forBRNC, MRNC, MpRNC, MbRNC, MsRNC, MapRNC and MvRNC.

It is to be understood that RNA may be isolated from gram-negative andgram-positive bacteria, and from mycobacteria, Mycobacterium phlei,Mycobacterium bovis BCG, Mycobacterium smegmatis, Mycobacterium aviumsubspecies paratuberculosis and Mycobacterium vaccae, and formulatedwith the chitosan nanoparticles as described in this example withoutadding the cell walls from these species.

It is to be realized that B16 melanoma cells are used in this example ofa typical cancer cell, and that chitosan nanoparticles are used in thisexample as a typical pharmaceutical delivery system for NA, and that oneskilled in the art will appreciate the applicability using such types ofpharmaceutical delivery systems and formulations in the treatment ofcancer in general.

Example 38 Preparation of Mycobacterium avium SubspeciesParatuberculosis RNA-Containing Composition (MapRNC)

In this example, intact Mycobacterium avium subspecies paratuberculosiscells are first washed by low-speed centrifugation to remove culturemedium components, and are then disrupted using high-pressurehomogenization with an Avestin EmulsiFlex-05 high-pressure homogenizer.After high-pressure homogenization the remaining intact mycobacteria areremoved by differential centrifugation using relative centrifugal forcesthat are optimized for the controlled removal of any residual, intactand undisrupted mycobacterium. The mycobacterial cell depleted fraction,comprising nucleic acids of the mycobacterium associated withmycobacterial cell wall fragments and the desired and controlled amountsof intact mycobacterial cells, is further purified by centrifugationwashing at a higher relative centrifugal force, to remove solublecontaminants. The mycobacterial cell-depleted fraction is isolated as apellet following centrifugation washing at high relative centrifugalforce. The mycobacterial cell-depleted fraction is then heat-treated at121° C. for between 5-30 min and used as mycobacterial RNC fromMycobacterium avium paratuberculosis (MapRNC).

Example 39 Analysis of Nucleic Acids in MapRNC

In this example the nucleic acid type, oligoribonucleotide chain lengthand content of MapRNC are determined. MapRNC is prepared as described inExample 38. Nucleic acids are extracted using the following procedure.An aliquot of 700 μL at a concentration of 1 mg/ml is digested withDNase- and RNase-free lysozyme, followed by inactivation and furtherdigestion with DNase- and RNase-free proteinase K (both fromSigma-Aldrich Canada, Oakville, Ontario). Nucleic acids are extracted byphenol/chloroform/isoamyl alcohol (25:24:1 v/v), and precipitated by theaddition of glycogen, sodium acetate and ethanol. The precipitates arewashed with 80% ice-cold ethanol, and resuspended in 50 μL distilledwater. The concentration is determined by measurement of the absorbanceat 260/280 nm in a UV spectrophotometer. The nucleic acid content ofeach composition is determined.

MapRNC preparations collected before and after heat-treatment (121° C.,30 min) are analyzed electrophoretically for their nucleic acid profileusing the Bioanalyzer system (Bioanalyzer model #2100, Agilent, SantaClara, Calif., USA). The nucleic acid fraction is diluted to aconcentration of 30 ng/μL. Electrophoretic analysis of the length of theextracted nucleic acid is accomplished with the Bioanalyzerelectrophoresis unit using the RNA 6000 nano kit (Agilent #5067-1511).This kit provides information on the quality of RNA in a size range offrom 25 to 6000 nucleotides. MapRNC following autoclaving is thenfurther analyzed using the Small RNA Kit (Agilent Technologies CanadaInc., St. Laurent, Québec, Canada, kit #5067-1548), which is designedfor the analysis of small nucleic acids in the size range of 6 to 150nucleotides. MapRNC prior to heat-treatment possesses a nucleic acidprofile of between approximately 25 bases and close to 4000 bases whenanalyzed using the RNA nano 6000 kit. The result demonstrates that theuse of high-pressure homogenization steps (different pressurization andnumber of cycles) to prepare MapRNC results in a composition thatcontains a polyribonucleotide chain length of 25-4000 bases. Followingheat-treatment, MapRNC shows a more compact distribution. MapRNCpossesses an oligoribonucleotide peak that is maximal between 20-40bases, and with a nucleic acid profile of between 5 and 60 bases inlength. The MapRNC compositions have only minor amounts of nucleic acideluting at 100 bases, and even less oligoribonucleotide material elutingat about 150 bases in length. The results demonstrate that the use ofhigh-pressure homogenization and heat treatment steps (differentpressurization and number of cycles along with autoclaving) to prepareMapRNC results in a composition that contains an oligoribonucleotide andpolyribonucleotide chain length of less than 60 bases.

Analysis of the DNA to RNA ratio of MapRNC prepared as in Example 38 isfirst performed using enzymatic digestion with RNase-A of the extractednucleic acids followed by Bioanalyzer 2100 electrophoresis profiling andoligoribonucleotide content quantification by using the Agilent SmallRNA Kit (kit #5067-1548). RNase-A digestion of the extracted nucleicacids is carried out using DNase-free Ribonuclease A (RNase A, treatedat 100° C. for 30 min to remove DNase activity) (0.1 μg enzyme, 2 h at37° C.). The RNase A is obtained from Ameresco (Solon, Ohio, USA). Asample (20 ng/μL) of RNase A-treated nucleic acid is analyzed using theBioanalyzer. The amount of DNA and RNA is determined in MapRNC using theequation:

DNA content=(Total Nucleic Acid Content−Nucleic Acid Content afterRNase-A treatment). The analysis of MapRNC shows that the presence ofDNA and RNA in the extract of MapRNC.

Analysis of the DNA to RNA ratio of MapRNC of the present invention isperformed as follows. The nucleic acid fraction is recovered byultrafiltration using a Microsep 1K unit (molecular weight cutoff=1000Da, Pall® Life Sciences, Ann Arbor, Mich., USA). The nucleic acidsolution is then digested to a mixture of nucleoside 5′-monophosphatesusing nuclease P1 (Sigma-Aldrich, Oakville, ON, Canada) following theprocedure reported by Liang (Liang et al., Ann. Chim. Acta 2009,650:106-110). To ensure optimal nuclease P1 digestion, a total of 50 μLof the nucleic acid aqueous solutions to be investigated are heated in awater bath at 95-100° C. for 10 min, followed by immediate chilling onice. Nuclease P1 is prepared at 5 units/μL in 30 mM sodium acetatebuffer containing 0.5 mM ZnCl₂, pH 5.3. For enzymatic digestion, 50 μLof nucleic acid in aqueous solution is mixed with the same volume ofnuclease P1 solution and then incubated at 50° C. for 30 min. Theresulting mixture is cooled to room temperature and filtered through aNanosep 10K filter by centrifugation at 10,000 g for 20 min at roomtemperature prior to HPLC analysis.

Serial dilutions of a mixture of mononucleotide standards(5′-deoxyribonucleotides and 5′-ribonucleotides, Sigma-Aldrich)containing 100, 10, 5, 2.5, 1 ng/μL each of the mononucleotide presentin DNA and RNA are also treated to nuclease P1 treatment and filteredfor use as standards in HPLC analysis. The elution order of thesenucleotides is confirmed by comparing the retention time of individualnucleotides under the same HPLC condition. HPLC analysis is performedusing a 1200 series HPLC system (Agilent, St-Laurent, Quebec, Canada),which is equipped with a quaternary pump with degasser, an autosampler,a column heater, and a multi-wavelength UV detector. A ZORBAX Bonus-RP(reverse phase) column (Agilent Technologies) is used and the mobilephases comprised a linear gradient of 10 mM potassium phosphate buffer,pH 7.2 and methanol (0-10% methanol). The mononucleotides are detectedat 260 nm. The DNA:RNA ratio is determined after determination of DNAand RNA content. The result demonstrates that RNA is present in MapRNCin addition to DNA.

Example 40 MapRNC Induces the Activation of the HumanPathogen-Associated Molecular Pattern Receptor (PAMP) NOD2

The NOD2 activation activity of MapRNC as prepared in Example 38 isevaluated using HEK-293 cells engineered to express the human NOD2receptor driving NF-κB and a downstream signalling marker IL-8.

The human HEK293-NOD2 cell line (InvivoGen, San Diego, Calif., USA) iscultured and maintained in high glucose DMEM, supplemented with 10%fetal bovine serum (both from Wisent, St-Bruno, Québec, Canada), 100μg/mL Normocin™ and 10 μg/mL blasticidin (both from InvivoGen) at 37° C.in a humidified atmosphere containing 5% CO₂. HEK293-NOD2 cells areseeded at 5×10⁵ cells/mL in a volume of 0.2 mL in sterile 96-wellflat-bottomed tissue culture microplates in the cell culture mediumdescribed above, and incubated for 48 hours with 0.625, 1.25, 2.5, 5,10, 20, 40, 80 and 160 μg/mL MapRNC at 37° C. in a humidified atmospherecontaining 5% CO₂. Supernatants are collected after incubation,centrifuged at 4,000×RCF for 5 min at 4° C. to remove cells and debrisand the supernatant is stored at −20° C. for analysis. Human IL-8 in thesupernatant is measured in the supernatants after 48 hours cultivationwith MapRNC by means of a commercial enzyme-linked immunosorbent assay(ELISA) from BioSource, Camarillo, Calif., USA. Data are captured froman ELISA plate reader (ELx-808IU Bio Tek Instruments, Winooski, Vt.,USA) using the KC Junior software package (Bio Tek Instruments,Winooski, Vt., USA) and expressed in pg/mL of IL-8 synthesized. MapRNCinduces the synthesis and excretion of IL-8 in a dose-dependent manner.The result therefore shows that MapRNC functions as a NOD2 agonist byinducing NOD2-driven IL-8 release in a dose-dependent manner.

Example 41 MapRNC has Human TLR2 Activation Activity

The TLR2 activation activity of MapRNC as prepared in Example 38 ismeasured using HEK-293 cells engineered to express the human TLR2receptor.

The human HEK293-TLR2 cell line is obtained from InvivoGen, San Diego,Calif., USA. The HEK293-TLR2 cells are stably transfected with the TLR2gene and a TLR2 receptor-driven secreted embryonic alkaline phosphatasegene (SEAP), placed under the control of the NF-kB gene. Activation ofTLR2 thus results in the generation of alkaline phosphate activity inthe cell culture medium, which is used to quantify receptor activation.The cells are cultured and maintained in high glucose DMEM, supplementedwith 10% fetal bovine serum (both from Wisent, St-Bruno, Québec, Canada)and 1× Normocin™ (InvivoGen) at 37° C. in a humidified atmospherecontaining 5% CO₂. HEK293-TLR2 cells are seeded at 5×10⁵ cells/mL in avolume of 0.2 mL in sterile 96-well flat-bottomed tissue culturemicroplates in HEK-Blue™ Detection medium (InvivoGen), and incubated for18 hours with 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 μg/mL MapRNCat 37° C. in a humidified atmosphere containing 5% CO₂. The HEK-Blue™Detection medium added to the wells is designed for the detection ofNF-kB induced soluble embryonic alkaline phosphatase enzyme activity(SEAP activity). After an 18 hour incubation, the optical density (whichis proportional to the activation of NF-kB through TLR2 engagement) isdetermined at 630 nm using a microplate spectrophotometer reader(ELx-808IU model, Bio Tek Instrument, Winooski, Vt., USA). Data iscaptured using the KC Junior software package (Bio Tek Instruments).SEAP activity in the cell supernatant is increased in a dose-relatedmanner following MapRNC treatment. The results therefore show thatMapRNC is capable of activating TLR2 in a dose-dependent manner (i.e.,MapRNC acts as a TLR2 agonist).

Example 42 Anti-Cancer Activity of MapRNC

The anticancer activity of MapRNC as prepared in Example 38 isdetermined using cancer cell lines representative of the major cancercell types. RT4 human bladder cancer cells (ATCC catalog # HTB-2™) areplated in DMEM culture medium and CT26 murine colon cancer cell lines(ATCC catalog # CRL2638™) in RPMI 1640 medium both supplemented with 10%heat-inactivated FBS (both from Wisent) and 10 μg/mL gentamycin(Sigma-Aldrich) at a concentration of 5×10⁵ cells/mL in 96-well tissueculture plates (50 μL volume), and allowed to adhere to the surface ofthe wells overnight at a temperature of 37° C. in an atmosphere of 5%CO₂ prior to treatment. Human leukemia cell Jurkat (ATCC TIB-152™) issuspended in DMEM culture medium supplemented with 10% heat-inactivatedFBS and 10 μg/mL gentamycin at a concentration of 1×10⁶ cells/mL in96-well tissue culture plates (50 μL volume). Heat-treated MapRNC (forexample 121° C., 30 min) in a volume of 50 μL tissue culture medium isadded to the cells to give final concentrations of 0.01, 0.1, 1.0 and 10μg/mL MapRNC. The cells are then incubated for 48 hours at 37° C. in anatmosphere of 5% CO₂. Anti-proliferative activity against the cancercell lines is determined using an MTT((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)reduction assay. Briefly, 10 μL of an MTT solution (5 mg/mL in PBS) isadded to each well after 48 hours incubation, and incubation continuesfor a further 4 hours. The reaction is then stopped by the addition of100 μL isopropanol:HCl (24:1 v/v). The reduction of MTT leads to theproduction of formazan. Formazan is solubilized by use of amicropipette, and the optical density (O.D.) determined in a microplatereader at a wavelength of 570 nm against appropriate controls. Thepercentage inhibition of cell proliferation is determined using theequation:

% Inhibition=100−[(Test O.D.−Control O.D.)/Control O.D.].

The results show that heat-treated MapRNC has dose-relatedanti-proliferative activity against RT4 bladder cancer cells and CT26colon cancer cells as well as Jurkat leukemic cells. MapRNC thereforehas anticancer activity that is not restricted to cancer type.

Example 43 Combination MRNC Formulations for Immune Stimulation

One of skill in the art after reading the above examples will use anappropriate combination of MRNC to achieve optimal therapeutic activityfor the desired indication. Thus, a combination of MpRNC and MvRNC willgive optimal NOD2 and TLR2 activation for immune stimulation andcytokine induction. Similarly, a combination of MpRNC and MvRNC willgive optimal anticancer activity and immune stimulation.

Example 44 Combination of MRNC with Therapeutic Agents Used in theTreatment of Cancer

MRNC compositions of the present invention are combined withchemotherapy and/or immunotherapy in order to enhance clinicaleffectiveness. The addition of MRNC to the following chemotherapeuticregimens is not intended to be a comprehensive listing (such listing areavailable in for example The Elsevier Guide to Oncology Drugs andRegimens, 2006, which describes more than 220 drug regimens commonly inthe treatment of cancer), and it is to be understood that those skilledin the art will, after reading the examples of the present invention,use the compositions of the present invention in the treatment of cancerin neoadjuvant or adjuvant settings with the most appropriatechemotherapy regimens. The following 2 protocols serve to illustrate theprinciple of how MRNC is used in combination with chemotherapy andimmunotherapy:

Colon and rectum cancers: Patients with colon and rectum cancers whohave locally advanced disease are at high risk for liver metastasesthrough hepatic portal vein seeding of the liver. Chemotherapy is ofteninitiated prior to or after surgical resection (neoadjuvant or adjuvantsettings) with the aim of reducing the incidence of hepatic metastasesand subsequent secondary metastasis to other organs. 5-flurouracil isoften used in the treatment of metastatic or advanced disease using aregimen of 1000 mg/m² by continuous IV infusion daily for 5 days whichis repeated every 28 days. MRNC of the present invention can be given asan intravenous bolus or infusion at a therapeutically active dose(determined through clinical evaluation studies) prior to, during orafter treatment with 5-fluorouracil. MRNC formulations may beadministered at a dose determined in preclinical testing and clinicalevaluations (phase-1, phase-2 and phase-3 clinical studies) to be themost effective. Such doses will be in the range 0.00001 mg/kg to about100 mg/kg per dose. Patients receiving combined therapy benefit fromincreased time to recurrence, decreased metastases, decreased tumorburden or eradication of tumor when compared to patients who receive5-fluorouracil therapy alone. Use may be made of other chemotherapyregimens known to those skilled in the art such as but not limited to5-fluorouracil, leucovorin and oxaliplatin with or without bevacizumab;irinotecan, leucovorin and 5-fluorouracil, with or without cetuximab; ormitomycin c and 5-fluorouracil, without limiting the effectiveness ofMRNC.

Lung cancer: Cyclophosphamide in combination with other chemotherapeuticagents is used for the treatment previously untreated patients, orpatients with advanced, recurrent or metastatic cancer. Typical regimensare but not limited to cyclophosphamide, doxorubicin (Adriamycin) andetoposide (CAE), where cyclophosphamide is administered at a dose of1000 mg/m² I.V. on day 1, doxorubicin is administered at a dose of 45mg/m² I.V. on day 1, and etoposide is administered at a dose of 100mg/m² I.V. on days 1 through 3. The treatment cycle is repeated every 28days. MRNC of the present invention is administered as an I.V. bolus atan optimal dose (determined MRNC formulations may be administered at adose determined in preclinical testing and clinical evaluations(phase-1, phase-2 and phase-3 clinical studies) to be the most effectivefor enhancing macrophage and granulocyte production through thestimulation of MU-CSF, G-CSF and GM-CSF either prior to, during or aftereach chemotherapeutic drug treatment cycle. Such doses will be in therange 0.00001 mg/kg to about 100 mg/kg per dose. Patients treated withMRNC have a reduced incidence of infectious episodes and delayedtreatment cycles related to chemotherapy-induced reductions in monocyteand neutrophil counts when compared to patients treated withchemotherapy alone. Use may be made of other chemotherapy regimens forthe treatment of lung cancer (primary or metastatic) without limitingthe effectiveness of MRNC.

Cervical cancer: Cervical cancer is associated with HPV infection.Although some 15 HPV types are oncogenic, HPV16 and 16 are believed tobe responsible for 70% of all cervical cancers. Current immunizationprotocols are effective in preventing infection, but are not effectivein treating established infections. There is an unmet need in thetreatment of cervical cancer where therapy against the cancer cells andan ongoing viral infection is required if cervical function is to bepreserved. Advanced, recurrent or metastatic disease is treated withcarboplatin 50-100 mg/m2 I.V. and doxorubicin 45-60 mg/m2 I.V. on day 1,and the cycle is repeated every 21 days. MRNC is administered locallyinto the cervix by intracervical injection at a dose determined to beoptimal for immune stimulation through previous clinical investigationprior to, during or after chemotherapy. MRNC formulations may beadministered at a dose determined in preclinical and clinicalevaluations (phase-1, phase-2 and phase-3 clinical studies) to be themost effective. Such doses will be in the range 0.00001 mg/kg to about100 mg/kg per dose. MRNC has 2 effects, first there is a reduction inviral load of HPV viruses (including HPV 16 and 18) in the cervixthrough the generation of an immune response against thevirally-infected cells via the induction of an immune response, andsecond, an augmentation of the clinical effectiveness of thechemotherapeutic agents through the induction of MU-CSF, G-CSF andGM-CSF.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to different embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

We claim:
 1. A composition comprising: mycobacterial RNA andmycobacterial cell walls, wherein the composition does not containphenol or pronase, and the mycobacterial RNA is in the form ofoligoribonucleotides and polyribonucleotides, wherein the length of theoligoribonucleotides and polyribonucleotides is about 2 to about 4000bases, about 2 to about 150 bases, about 20 to about 40 bases, or about2 to about 40 bases.